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 Babraham Institute

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.

L&#39;extr&eacute;mit&eacute; amin&eacute;e de la phospholipase D1 (PLD1) contient trois d&eacute;terminants potentiels de membrane-interaction : un domaine d&#39;homologie (PX) phox, un domaine d&#39;homologie (PH) de pleckstrine et deux cyst&eacute;ines adjacentes aux positions 240 et 241 dans le domaine de PH qui sont gras acyl&eacute;s in vivo. Pour comprendre comment ces facteurs contribuent &agrave; la localisation de la membrane, nous avons mutag&eacute;nis&eacute;es tant des r&eacute;sidus importants du domaine PLD1 PH dans le type sauvage ou exempt de palmitate de fond dans la prot&eacute;ine intacte, dans un fragment qui supprime les premier 210 acides amin&eacute;s dont le domaine PX et dans le domaine de PH isol&eacute;. Mutants ont &eacute;t&eacute; exprim&eacute;s dans des cellules COS-7 et examin&eacute;s pour r&eacute;sidence de membrane, de localisation intracellulaire, palmitoylation et activit&eacute; catalytique. Nos r&eacute;sultats sont comme suit. 1) Mutag&eacute;n&egrave;se de r&eacute;sidus importants du domaine PH se traduit par la redistribution de PLD1 des membranes au cytosol, ind&eacute;pendamment des sites acylation gras. Ce qui est important, des mutants de domaine de PH dans le fond de type sauvage a montr&eacute; une acylation grasse fortement r&eacute;duite, malgr&eacute; la pr&eacute;sence de tous les cyst&eacute;ines pertinentes. 2) Le domaine de PH isol&eacute; ne pas colocalis&eacute;s avec PLD1 et n&#39;a pas &eacute;t&eacute; palmitoyl&eacute;es. 3) Mutant suppression PX a montr&eacute; similaire de distribution et de palmitoylation &agrave; la prot&eacute;ine intacte. Fait int&eacute;ressant, des mutants de domaine de PH dans ce contexte a montr&eacute; palmitoylation significative et redistribution cytosolique incompl&egrave;te. 4) Mutants de domaine PH dans le type sauvage ou exempt de palmitate de fond a maintenu l&#39;activit&eacute; catalytique. Nous proposons que membrane ciblage de PLD1 implique une hi&eacute;rarchie des signaux avec un domaine de PH fonctionnel, ce qui permet d&#39;acylation grasse menant &agrave; liaison forte membrane. Le domaine de la PX peut moduler la fonction du domaine PH.

Hi&eacute;rarchie des signaux de ciblage membranaire de la phospholipase D1 impliquant la modification de lipide d&#39;un domaine d&#39;homologie de pleckstrine.

Il terminus amminico della fosfolipasi D1 (PLD1) contiene tre determinanti di membrana-interazione potenziale: un dominio di omologia (PX) phox, un dominio di omologia (PH) di settori e due cisteine adiacenti alle posizioni 240 e 241 all&#39;interno del dominio di PH che sono grasso acilata in vivo. Per capire come questi fattori contribuiscono alla localizzazione di membrana, noi abbiamo mutagenized critici residui del dominio PH PLD1 nel tipo selvatico o palmitato senza sfondo della proteina intatta, in un frammento che elimina i primo 210 amminoacidi compreso il dominio PX e nel dominio PH isolato. Mutanti erano espresse in cellule COS-7 ed esaminati per residenza di membrana, localizzazione intracellulare, palmitoilazione e attivit&agrave; catalitica. I nostri risultati sono come segue. 1) Mutagenesi di residui critici del dominio PH risultati nella ridistribuzione di PLD1 dalle membrane al citosol, indipendentemente dai siti acilazione grassi. Soprattutto, mutanti dominio PH sullo sfondo di tipo selvatico ha mostrato acilazione grassi notevolmente ridotto, nonostante la presenza di tutte le cisteine pertinenti. 2) Il dominio di PH isolato non co-localize con PLD1 e non era palmitoilazione. 3) Il mutante di delezione PX ha mostrato simile distribuzione e palmitoilazione alla proteina intatta. &Egrave; interessante notare, mutanti dominio PH in questo sfondo ha mostrato significativi palmitoilazione e ridistribuzione citosolico incompleta. 4) Mutanti dominio PH nel tipo selvatico o sfondo libero palmitato mantenuto attivit&agrave; catalitica. Proponiamo che la membrana di targeting di PLD1 comporta una gerarchia dei segnali con un dominio PH funzionale permettendo acilazione grassi che porta membrana forte legame. Il dominio PX possa modulare la funzione del dominio PH.

Gerarchia dei segnali di membrana-targeting della fosfolipasi D1 che comportano modificazione dei lipidi di un dominio di omologia di settori.

Chinasi sfingosina 1 (SK1) fosforila sfingosina per generare sfingosina 1-fosfato (S1P). Perch&eacute; il prodotto dell&#39;enzima e substrato sono potenzialmente importanti molecole, il regolamento di SK1 di segnalazione &egrave; di notevole interesse. Segnaliamo che SK1, che normalmente &egrave; un enzima citosolico, trasloca in vivo e in vitro a compartimenti membrana arricchito in acido fosfatidico (PA), il prodotto di lipidi della fosfolipasi D. Questa traslocazione dipende dall&#39;interazione diretta di SK1 con PA, perch&eacute; enzima ricombinante purificato Mostra forte affinit&agrave; per la PA puro accoppiato ad Affi-Gel. L&#39;interazione SK1-PA le mappe per il C-terminale di SK1 ed &egrave; indipendente dell&#39;attivit&agrave; catalitica o del diacilglicerolo chinasi-come dominio dell&#39;enzima. Cos&igrave; SK1 costituisce un romanzo, fisiologicamente rilevanti effettrici PA.

Chinasi 1 della sfingosina &egrave; un effector intracellulare di acido fosfatidico.

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.

Kinase de sphingosine 1 (SK1) phosphoryle sphingosine pour g&eacute;n&eacute;rer la sphingosine 1-phosphate (S1P). Comme substrat et produit de l&#39;enzyme sont potentiellement importants signalisation des mol&eacute;cules, la r&eacute;glementation des SK1 est d&#39;un int&eacute;r&ecirc;t consid&eacute;rable. Les auteurs rapportent que SK1, qui est ordinairement une enzyme cytosolique, translocation in vivo et in vitro aux compartiments membranaires enrichis en acide phosphatidique (PA), le produit lipidique de la phospholipase D. Cette translocation d&eacute;pend de l&#39;interaction directe de SK1 avec PA, parce que l&#39;enzyme purifi&eacute;e recombinante montre une affinit&eacute; forte pour pure PA coupl&eacute; &agrave; Affi-Gel. L&#39;interaction SK1-PA correspond &agrave; la partie C terminale de SK1 et est ind&eacute;pendante de l&#39;activit&eacute; catalytique ou du domaine kinase-comme de l&#39;enzyme diacylglyc&eacute;rol. SK1 constitue donc un roman, physiologiquement pertinent effecteur de PA.

Sphingosine kinase 1 est un effecteur intracellulaire de l&#39;acide phosphatidique.

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.

RhoG est une famille Rho que GTPase petit impliqu&eacute; dans la r&eacute;gulation du cytosquelette, agissant soit en amont de, ou en parall&egrave;le de Rac1. L&#39;ou les fonctions pr&eacute;cise de RhoG in vivo n&#39;a pas encore &eacute;t&eacute; d&eacute;finie. Nous avons identifi&eacute; un nouveau r&ocirc;le pour RhoG dans la signalisation l&#39;&eacute;clat respiratoire neutrophil stimul&eacute; par les agonistes des r&eacute;cepteurs coupl&eacute;s aux prot&eacute;ines G. Moelle osseuse neutrophiles de RhoG knockout (RhoG(-/-)) souris pr&eacute;sentaient une d&eacute;ficience marqu&eacute;e de la g&eacute;n&eacute;ration d&#39;oxydant en r&eacute;ponse &agrave; C5a ou fMLP, mais des r&eacute;ponses normales aux PMA ou zymosan opsonis&eacute;e et mort bact&eacute;rienne normale. Activation de Rac1 et Rac2 par fMLP a diminu&eacute; neutrophiles RhoG(-/-) seulement &agrave; tr&egrave;s t&ocirc;t (5 s) fois points (de 25 % et 32 % respectivement), tandis que la chimiotaxie en r&eacute;ponse aux agonistes solubles n&#39;est pas affect&eacute;e par le manque de RhoG. En outre, fMLP stimul&eacute;e par la phosphorylation de la prot&eacute;ine kinase B et p38MAPK, l&#39;activation de la phospholipase D et flux de calcium &eacute;taient &eacute;quivalents dans le type sauvage et RhoG(-/-) neutrophiles. Nos r&eacute;sultats d&eacute;finissent RhoG comme un &eacute;l&eacute;ment essentiel de la G protein-coupled stimul&eacute;e par le r&eacute;cepteur cascades de signalisation dans les neutrophiles murines, agissant soit via un sous-ensemble de cellulaire Rac encore ind&eacute;fini et pertinent &agrave; l&#39;activation de l&#39;oxydase et/ou d&#39;une nouvelle interaction totale avec le neutrophil NADPH oxydase.

RhoG r&eacute;glemente le neutrophil NADPH oxydase.

RhoG &egrave; una famiglia di Rho che piccola GTPasi implicata nella regolazione del citoscheletro, agisca a monte di o in parallelo a Rac1. La funzione precisa di RhoG in vivo non &egrave; stato ancora definito. Abbiamo individuato un nuovo ruolo per RhoG nella segnalazione il burst respiratorio dei neutrofili stimolato dagli agonisti dei recettori accoppiati a proteine G. Neutrofili derivate dal midollo osseo dal RhoG ko (RhoG(-/-)) topi esposto una compromissione marcata della generazione ossidante in risposta a C5a o fMLP, ma normali risposte alla PMA o opsonized zymosan e uccisione batterica normale. Attivazione di Rac1 e Rac2 da fMLP era diminuito in RhoG(-/-) neutrofili solo a molto presto (5 s) punti di tempo (da 25 e 32%, rispettivamente), mentre la chemiotassi in risposta agli agonisti solubili &egrave; stata influenzata dalla mancanza di RhoG. Inoltre, stimolato fMLP fosforilazione della chinasi di proteina B e p38MAPK, attivazione della fosfolipasi D e flussi di calcio erano equivalenti nel wild-type e RhoG(-/-) neutrofili. I nostri risultati definiscono RhoG come una componente critica di G protein-coupled recettore stimolato segnalazione cascate in neutrofili murini, agendo tramite un sottoinsieme della cellular Rac pertinente all&#39;attivazione di ossidasi e/o di un romanzo e non ancora definito interazione totale con il NADPH ossidasi dei neutrofili.

RhoG regola i neutrofili NADPH ossidasi.

PA binding of phosphatidylinositol 4-phosphate 5-kinase.

PA contraignant du phosphatidylinositol 4-phosphate 5-kinase.

PA associazione di fosfatidilinositolo 4-fosfato 5-chinasi.

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.

Autophagie est l&#39;engouffrement du cytosol et organites par double membrane des v&eacute;sicules appel&eacute; autophagosomes. Formation de l&#39;autophagosome est connue pour exiger la phosphatidylinositol 3-phosphate (PI(3)P) et se trouve pr&egrave;s de r&eacute;ticulum endoplasmique (re), mais l&#39;exact m&eacute;canismes sont inconnus. Nous montrons que la prot&eacute;ine double de domaine contenant des FYVE 1, une prot&eacute;ine de liaison (3) P PI avec localisation inhabituelle sur les membranes ER et Golgi, translocation en r&eacute;ponse &agrave; la famine d&#39;acides amin&eacute;s &agrave; un compartiment ponctu&eacute; partiellement colocalis&eacute; avec des prot&eacute;ines d&#39;autophagosomal. La translocation est tributaire de la fonction Vps34 et beclin. Autres sondes de liaison (3) P PI cibl&eacute;s au re montrent la m&ecirc;me translocation d&#39;induite par le je&ucirc;ne qui est tributaire de la reconnaissance et la formation de PI (3) P. Des exp&eacute;riences d&#39;imagerie Live montrent que ce compartiment ponctu&eacute; forme pr&egrave;s de v&eacute;sicules contenant du Vps34, est en &eacute;quilibre dynamique avec l&#39;ER et fournit une plateforme de membrane pour l&#39;accumulation de prot&eacute;ines autophagosomal, expansion des membranes autophagosomal et &eacute;mergence des autophagosomes enti&egrave;rement form&eacute;es. Ce compartiment P-enrichi de PI (3) peut-&ecirc;tre &ecirc;tre impliqu&eacute; dans la biogen&egrave;se autophagosome. Sa relation dynamique avec l&#39;ER est conforme &agrave; l&#39;id&eacute;e que l&#39;ER peut fournir des &eacute;l&eacute;ments importants pour la formation de l&#39;autophagosome.

Formation de l&#39;autophagosome des compartiments membranaires enrichis en phosphatidylinositol 3-phosphate et connect&eacute;s dynamiquement vers le r&eacute;ticulum endoplasmique.

Autophagy &egrave; il engulfment del citosol e organelli da doppia membrana vescicole denominate autophagosomes. &Egrave; noto che Autophagosome formazione richiedono fosfatidilinositolo-3-fosfato (PI(3)P) e si verifica in prossimit&agrave; del reticolo endoplasmatico (ER), ma l&#39;esatto meccanismi sono sconosciuti. Mostriamo che la doppia FYVE dominio contenenti proteine 1, una PI (3) P-proteina con insolita localizzazione su membrane ER e Golgi, trasloca in risposta alla fame di aminoacidi per un vano punctato parzialmente colocalized con proteine autophagosomal. Traslocazione dipende dalla funzione Vps34 e Ambra1. Altre sonde (3) P-associazione PI mirati al ER mostrano la stessa traslocazione indotta da fame che dipende dal riconoscimento e formazione di PI (3) P. Live imaging esperimenti mostrano che questo vano punctato fa vicino le vescicole contenenti Vps34, &egrave; in equilibrio dinamico con l&#39;ER e fornisce una piattaforma di membrana per accumulo di proteine autophagosomal, espansione delle membrane autophagosomal e comparsa di autophagosomes completamente formata. Questo vano P-arricchito di PI (3) possa essere coinvolti nella biogenesi autophagosome. Suo rapporto dinamico con l&#39;ER &egrave; coerenza con l&#39;idea che l&#39;ER pu&ograve; fornire componenti importanti per la formazione di autophagosome.

Autophagosome formazione da membrana compartimenti arricchito in fosfatidilinositolo-3-fosfato e dinamicamente collegati al reticolo endoplasmatico.

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.

Autophagie pr&eacute;sente un d&eacute;fi topologique pour la cellule, car elle exige la livraison de mat&eacute;riel cytosolique &agrave; la lumi&egrave;re d&#39;un compartiment membranaire, le lysosome. Ceci a &eacute;t&eacute; r&eacute;solu d&#39;une mani&egrave;re ing&eacute;nieuse par la formation d&#39;une v&eacute;sicule double membrane, l&#39;autophagosome, qui capte des prot&eacute;ines cytosoliques et organites pendant sa transformation d&#39;un disque de membrane plane en une sph&egrave;re. De cette fa&ccedil;on, cytosolique mat&eacute;riel, tout d&#39;abord, devient lumi&egrave;re et est assur&eacute;e pour d&eacute;gradation du lysosome. Une s&eacute;rie non r&eacute;solue des questions dans l&#39;autophagie concernant la membrane de l&#39;autophagosome : Quels sont les signaux pour sa formation et quelle est son identit&eacute; ? R&eacute;cemment, nous avons fourni quelques indices qui peuvent aider &agrave; r&eacute;pondre &agrave; ces questions. En suivant la dynamique de plusieurs phosphatidylinositol 3-phosphate (PI3P)-prot&eacute;ines de liaison au cours des acides amin&eacute;s (et l&#39;induction de l&#39;autophagie) nous avons conclu qu&#39;au moins certains autophagosomes se forment dans un compartiment de membrane induite par le je&ucirc;ne, enrichie en PI3P dynamiquement reli&eacute; au r&eacute;ticulum endoplasmique (re). Nous avons appel&eacute; les membranes de ce compartiment d&#39;omegasomes (&agrave; partir de leur forme om&eacute;ga). Nos r&eacute;sultats sugg&egrave;rent que la PI3P est important de fournir des indices de localisation et peut-&ecirc;tre pour faciliter l&#39;&eacute;tape de fusion &agrave; la phase finale de formation de l&#39;autophagosome.

Faire des autophagosomes : localis&eacute;e &agrave; la synth&egrave;se des cales de phosphatidylinositol 3-phosphate l&#39;indice.

Autophagy presenta una sfida topologica per la cella, poich&eacute; richiede la consegna di materiale citosolico al lume di un vano di membrana-limiti, il lisosoma. Questo &egrave; risolto in un modo ingegnoso per la formazione di una doppia membrana della vescicola, il autophagosome, che cattura le proteine citosoliche e organelli durante la sua trasformazione da un disco di membrana planare in una sfera. In questo modo, materiale citosolico prima diventa lumenal e poi viene consegnato per la degradazione per il lisosoma. Un set irrisolto di domande in autophagy interessa la membrana della autophagosome: che cosa sono i segnali per la sua formazione e che cosa &egrave; l&#39;identit&agrave;? Recentemente abbiamo fornito alcuni indizi che possono aiutare la risposta queste domande. Seguendo la dinamica dei diversi fosfatidilinositolo-3-fosfato (PI3P)-proteine di legame durante la fame dell&#39;amminoacido (e induzione autophagy) abbiamo concluso che almeno alcuni autophagosomes sono formate in un vano di membrana indotta da inedia, arricchita di PI3P dinamicamente collegati al reticolo endoplasmatico (ER). Abbiamo definito le membrane di questo vano omegasomes (dalla loro forma omega). I nostri dati suggeriscono che la PI3P &egrave; importante per fornire indizi di localizzazione e forse per agevolare la fase di fusione nella fase finale di formazione autophagosome.

Fare autophagosomes: localizzata sintesi delle stive di fosfatidilinositolo-3-fosfato l&#39;indizio.

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).

Catalyse de la phospholipase D (PLD) l&#39;hydrolyse de la phosphatidylcholine pour g&eacute;n&eacute;rer de l&#39;acide phosphatidique et choline. Historiquement, beaucoup de travail PLD a &eacute;t&eacute; r&eacute;alis&eacute;e dans param&egrave;tres chez les mammif&egrave;res, bien que les g&egrave;nes codant des enzymes de cette famille ont &eacute;t&eacute; identifi&eacute;s dans tous les organismes eucaryotes. R&eacute;cemment, des renseignements importants sur la fonction PLD &eacute;mergent du travail chez la levure, mais est beaucoup moins connu sur PLD chez d&#39;autres organismes. Dans cette revue, nous r&eacute;sumerons que sait-on de phospholipase D dans plusieurs organismes mod&egrave;les, y compris le c. elegans, d. discoideum, d. rerio et d. melanogaster. Dans les cas o&ugrave; les prises sont disponibles (c. elegans, Dictyostelium et Drosophila) l&#39;ou les g&egrave;nes PLD ne semblent ne pas &ecirc;tre essentiel pour la viabilit&eacute;, mais plusieurs &eacute;tudes commencent &agrave; identifier les voies o&ugrave; cette activit&eacute; a un r&ocirc;le. &Eacute;tant donn&eacute; que les prot&eacute;ines dans les organismes mod&egrave;les sont tr&egrave;s semblables &agrave; leurs homologues chez les mammif&egrave;res, nous attendons que les &eacute;tudes futures dans les organismes mod&egrave;les compl&eacute;teront et poursuivent leurs travaux en cours dans les param&egrave;tres de mammif&egrave;res. &Agrave; la fin de cet examen, nous fournirons &eacute;galement une br&egrave;ve mise &agrave; jour sur des cibles d&#39;acides phosphatidiques, un sujet Contenu revu en 2006.

Nouveaux r&eacute;sultats d&#39;&eacute;tudes de la phospholipase D organismes mod&egrave;les (et une br&egrave;ve mise &agrave; jour sur les effecteurs acides phosphatidiques).

Catalizza fosfolipasi D (PLD) l&#39;idrolisi della fosfatidilcolina per generare acido fosfatidico e colina. Storicamente, molto lavoro PLD &egrave; stata condotta nelle impostazioni dei mammiferi, anche se i geni che codificano enzimi di questa famiglia sono stati identificati in tutti gli organismi eucarioti. Recentemente, importanti approfondimenti sulla funzione PLD stanno emergendo dal lavoro in lievito, ma molto meno &egrave; conosciuto circa PLD in altri organismi. In questa recensione riassumiamo ci&ograve; che &egrave; noto circa fosfolipasi D in vari organismi modello, tra cui c. elegans, d. discoideum, d. rerio e d. melanogaster. Nei casi dove knockouts sono disponibili (c. elegans, Dictyostelium e Drosophila) i PLD gene (i) non sembrano essere essenziali per la vitalit&agrave;, ma diversi studi stanno cominciando a individuare percorsi dove questa attivit&agrave; ha un ruolo. Dato che le proteine in organismi modello sono molto simili alle loro controparti nei mammiferi, ci aspettiamo che gli studi futuri in organismi modello completer&agrave; ed estendere i lavori in corso nelle impostazioni dei mammiferi. Alla fine di questa recensione vi forniremo anche un breve aggiornamento sui bersagli acidi fosfatidico, un argomento ultima revisione nel 2006.

Risultanze emergenti dagli studi della fosfolipasi D in organismi modello (e un breve aggiornamento su effettori acidi fosfatidico).

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.

L&#39;acide phosphatidique (PA) &eacute;met l&#39;hypoth&egrave;se d&#39;avoir des fonctions structurelles et signalisation pendant la dynamique de la membrane dans les cellules animales. Dans cette &eacute;tude, nous montrons qu&#39;avant un d&eacute;lai critique au cours de la biogen&egrave;se des rhabdomere dans les photor&eacute;cepteurs de Drosophila melanogaster, des niveaux &eacute;lev&eacute;s de PA perturbent le transport membranaire au domaine apical. Lipidomic analyse montre que cet effet est associ&eacute; &agrave; une augmentation de l&#39;abondance d&#39;une seule esp&egrave;ce mol&eacute;culaire relativement mineure de Pennsylvanie. Ces d&eacute;fauts de transport d&eacute;pendent de l&#39;&eacute;tat d&#39;activation de Arf1. D&eacute;fauts de transport via PA g&eacute;n&eacute;r&eacute;e par la phospholipase D demander &agrave; l&#39;activit&eacute; de la kinase 4 phosphate 5 phosphatidylinositol (PI) de type I, sont ph&eacute;nocopies par pr&eacute;cipitation de la kinase 4 PI et sont associ&eacute;s avec le r&eacute;ticulum endoplasmique normal pour le transport de l&#39;appareil de Golgi. Nous proposons que les niveaux de PA sont critiques pour les &eacute;v&eacute;nements de transport de membrane apicale requis pour rhabdomere biogen&egrave;se.

Biogen&egrave;se des Rhabdomere dans les photor&eacute;cepteurs de la drosophile est tr&egrave;s sensible aux taux d&#39;acide phosphatidique.

Acido fosfatidico (PA) &egrave; postulato per avere funzioni strutturali e di segnalazione durante le dinamiche di membrana in cellule animali. In questo studio, mostriamo che prima di un periodo di tempo critico durante il rabdomero contiene biogenesi in fotorecettori melanogaster della drosofila, livelli elevati di PA perturbare trasporto di membrana al dominio apicale. Analisi lipidomica dimostra che questo effetto &egrave; associato con un aumento nell&#39;abbondanza di una singola specie molecolare relativamente minore di PA Questi difetti di trasporto dipendono lo stato di attivazione del Arf1. Difetti di trasporto via PA generato dalla fosfolipasi D imporre l&#39;attivit&agrave; della chinasi 4 fosfato 5 fosfatidilinositolo (PI) di tipo I, sono phenocopied di atterramento di chinasi PI 4 e sono associati con normale reticolo endoplasmico di massima per il trasporto di Golgi. Proponiamo che i livelli di PA sono critici per gli eventi di trasporto di membrana apicale necessari per rabdomero contiene biogenesi.

Biogenesi rabdomero contiene in fotorecettori drosofila sono acutamente sensibile a livelli di acidi fosfatidico.

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.

Autophagie est une r&eacute;ponse cellulaire &agrave; la famine qui g&eacute;n&egrave;re des autophagosomes pour transporter les organites cellulaires et prot&eacute;ines de longue dur&eacute;es vers les lysosomes pour d&eacute;gradation. D&eacute;gradation par autophagie peut fournir une d&eacute;fense inn&eacute;e contre l&#39;infection par le virus, ou inversement autophagosomes peut favoriser l&#39;infection par permettant l&#39;assemblage des prot&eacute;ines r&eacute;plicase. Nous d&eacute;montrons que le coronavirus aviaire, le Virus de la bronchite infectieuse (IBV) active autophagie. Un &eacute;cran de diff&eacute;rentes prot&eacute;ines non structurales IBV (NSP) a montr&eacute; que l&#39;autophagie a &eacute;t&eacute; activ&eacute; par IBV nsp6. Cette propri&eacute;t&eacute; a &eacute;t&eacute; partag&eacute;e avec nsp6 du Virus de l&#39;h&eacute;patite de souris, les coronavirus chez les mammif&egrave;res et l&#39;&eacute;quivalent nsp5-7 de la Nidovirales dysg&eacute;n&eacute;sique et respiratoire Syndrome Virus Virus du Syndrome respiratoire aigu s&eacute;v&egrave;re. Ces prot&eacute;ines transmembranaires traversant multiple situ&eacute;s vers le r&eacute;ticulum endoplasmique (ER) o&ugrave; ils ont g&eacute;n&eacute;r&eacute; des v&eacute;sicules Atg5 et LC3II positif, et la formation de v&eacute;sicules d&eacute;pendait de Atg5 et classe III PI3 kinase. Les v&eacute;sicules recrutement double FYVE-domaine contenant des prot&eacute;ines (DFCP) ce qui indique une concentration localis&eacute;e de phosphatidylinositol 3 phosphate et par cons&eacute;quent partagent de nombreuses caract&eacute;ristiques avec omegasomes form&eacute; &agrave; partir de la salle d&#39;urgence en r&eacute;ponse &agrave; la famine. Omegasomes induite par la nsp6 virale m&ucirc;ri dans autophagosomes LC3 vers les lysosomes et donc recrut&eacute; et recycl&eacute; les prot&eacute;ines n&eacute;cessaires &agrave; la nucl&eacute;ation autophagosome, expansion, trafic cellulaire et livraison de la cargaison vers les lysosomes. Les coronavirus nsp6 prot&eacute;ines activ&eacute;es formation omegasome et autophagosome ind&eacute;pendamment de famine, mais activation ne comportait pas d&#39;inhibition directe de mTOR, signalisation, activation de la sirtuin1 ou l&#39;induction du stress du re.

Coronavirus nsp6 prot&eacute;ines g&eacute;n&egrave;rent des autophagosomes du r&eacute;ticulum endoplasmique via un interm&eacute;diaire omegasome.

Autophagy &egrave; una risposta cellulare alla fame che genera autophagosomes per trasportare gli organelli cellulari e lisosomi per la degradazione delle proteine longeve. Degradazione attraverso autophagy in grado di fornire una difesa innata contro l&#39;infezione da virus, o al contrario autophagosomes pu&ograve; promuovere infezione facilitando l&#39;assemblaggio delle proteine replicasi. Dimostriamo che il coronavirus aviaria, Virus di bronchite infettiva (IBV) attiva autophagy. Una schermata di singole proteine non strutturali IBV (NSP) ha mostrato che autophagy &egrave; stato attivato di nsp6 IBV. Questa propriet&agrave; &egrave; stata condivisa con nsp6 di mammifero coronavirus Virus dell&#39;epatite del Mouse e Virus della sindrome respiratoria acuta grave e l&#39;equivalente nsp5-7 di arterivirus Virus della sindrome respiratoria e riproduttiva suina. Queste multiple spanning proteine transmembrana che si trova il reticolo endoplasmatico (ER) dove hanno generato le vescicole Atg5 e LC3II-positivi, e formazione di vescicola dipendeva dal Atg5 e classe III PI3 chinasi. Le vescicole reclutarono doppia FYVE-dominio contenenti proteine (DFCP) che indica la concentrazione localizzata del fosfatidilinositolo 3 fosfato e pertanto condiviso molte caratteristiche con omegasomes formata da ER in risposta alla fame. Omegasomes indotta da virale nsp6 maturata in autophagosomes che consegna LC3 ai lisosomi e quindi reclutato e riciclate le proteine necessarie per nucleazione autophagosome, espansione, traffico cellulare e consegna delle merci ai lisosomi. Le proteine nsp6 coronavirus attivato omegasome e autophagosome formazione indipendentemente dalla fame, ma non coinvolgono attivazione diretta inibizione di mTOR segnalazione, attivazione di sirtuin1 o induzione di stress ER.

Coronavirus nsp6 proteine generano autophagosomes dal reticolo endoplasmatico mediante un intermedio di omegasome.

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.

Les phosphoinositides simple PtdIns3P a &eacute;t&eacute; d&eacute;montr&eacute; pour contr&ocirc;ler la croissance des cellules en aval de la signalisation des acides amin&eacute;s et autophagie en aval du retrait de l&#39;acide amin&eacute;. Ces effets oppos&eacute;s d&eacute;pendent en partie de l&#39;existence de complexes distincts de Vps34 (prot&eacute;ine vacuolaire triant 34), la responsable de la majorit&eacute; de la synth&egrave;se de la PtdIns3P dans les cellules de kinase : un complexe unique est activ&eacute; apr&egrave;s le retrait de l&#39;acide amin&eacute; pour induire l&#39;autophagie et autre r&eacute;glemente activation (mammalian target of rapamycin complex 1) mTORC1 en pr&eacute;sence d&#39;acides amin&eacute;s. Cependant, signalisation de lipide-d&eacute;pendante presque toujours pr&eacute;sente une dimension spatiale, li&eacute;e au site de formation du signal lipidique. Dans le cas de l&#39;induction de l&#39;autophagie PtdIns3P r&eacute;glement&eacute;s, des donn&eacute;es r&eacute;centes sugg&egrave;rent que les PtdIns3P s&#39;accumule dans un compartiment membranaire connect&eacute; dynamiquement vers le r&eacute;ticulum endoplasmique qui constitue une plate-forme pour la formation de certains autophagosomes. Pour les activit&eacute;s r&eacute;glement&eacute;es PtdIns3P mTORC1, un contexte spatial n&#39;est pas encore connu : plusieurs possibilit&eacute;s peuvent se pr&eacute;senter &agrave; l&#39;issu des effets connus de PtdIns3P sur le syst&egrave;me d&#39;endocytose et les donn&eacute;es r&eacute;centes sugg&eacute;rant que l&#39;activation de mTORC1 d&eacute;pend de sa localisation sur les lysosomes.

Comment phosphoinositide 3-phosphate contr&ocirc;le la croissance en aval des acides amin&eacute;s et autophagie en aval du retrait de l&#39;acide amin&eacute;.

Il semplice phosphoinositide PtdIns3P ha dimostrato di controllare la crescita delle cellule a valle della segnalazione dell&#39;amminoacido e autophagy a valle del ritiro dell&#39;amminoacido. Questi opposti effetti dipendono in parte l&#39;esistenza di distinti complessi di Vps34 (Proteina vacuolar 34), il responsabile per la maggior parte della sintesi di PtdIns3P nelle cellule della chinasi: un unico complesso &egrave; attivato dopo il ritiro dell&#39;amminoacido per indurre autophagy e un&#39;altra regola mTORC1 attivazione (mammiferi bersaglio della rapamicina complesso 1) quando gli amminoacidi sono presenti. Tuttavia, quasi sempre segnalazione del lipido-dipendente esibisce una dimensione spaziale, correlata al sito della formazione del segnale lipidico. Nel caso di induzione PtdIns3P regolamentati autophagy, dati recenti suggeriscono che il PtdIns3P si accumula in un vano di membrana dinamicamente collegato a reticolo endoplasmatico che costituisce una piattaforma per la formazione di alcuni autophagosomes. Per attivit&agrave; di mTORC1 PtdIns3P-regolato, un contesto spaziale non &egrave; ancora noto: diverse possibilit&agrave; si possono prevedere basato sugli effetti conosciuti di PtdIns3P sul sistema endocitico e sui recenti dati suggeriscono che attivazione di mTORC1 dipende dalla sua localizzazione su lisosomi.

Come fosfoinositide 3-fosfato controlla la crescita a valle di aminoacidi e autophagy a valle del ritiro dell&#39;amminoacido.

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.

Autophagie basal-ici d&eacute;finie comme activit&eacute; macroautophagic au cours de la croissance cellulaire dans un milieu normal contenant des acides amin&eacute;s et s&eacute;rum-semble &ecirc;tre tr&egrave;s active dans de nombreux types de cellules et de tissus d&#39;origine animales. Ici, nous avons caract&eacute;ris&eacute; cette voie dans les cellules HEK 293 mammif&egrave;res. Tout d&#39;abord, nous avons examin&eacute;, c&ocirc;te &agrave; c&ocirc;te, trois compos&eacute;s qui sont employ&eacute;s couramment pour r&eacute;v&eacute;ler l&#39;autophagie basale en bloquant la maturation des autophagosomes : bafilomycines A 1 (BafA1), la chloroquine et la vinblastine. Seulement BafA1 s&#39;est av&eacute;r&eacute; sans compliquer les effets secondaires. La chloroquine partiellement inhib&eacute;e m&eacute;caniste cible de la rapamycine (MTOR) activit&eacute;, ce qui pourrait induire l&#39;induction de l&#39;autophagie ainsi que bloquer la maturation de l&#39;autophagosome. Vinblastine caus&eacute; la distribution des composants initiaux d&#39;omegasome en phagophore ponctu&eacute;e sites d&#39;assemblage, et donc il induirait &eacute;galement autophagie, compliquer l&#39;interpr&eacute;tation. Autophagie basale &eacute;tait nettement sensible &agrave; l&#39;inhibition par la wortmannine et par cons&eacute;quent requis formation du phosphatidylinositol 3-phosphate (PtdIns3P), mais c&#39;est deux fois aussi r&eacute;sistant aux wortmannin comme autophagie induite par la famine. Nous avons &eacute;galement d&eacute;termin&eacute; que l&#39;autophagie basale a &eacute;t&eacute; significativement supprim&eacute;e par activation de MTOR provoqu&eacute;e par la surexpression de RHEB ou chiffons activ&eacute;s. Enfin, nous avons &eacute;tudi&eacute; la relation spatiale des autophagosomes naissant dans le r&eacute;ticulum endoplasmique (ER) ou aux mitochondries par des exp&eacute;riences d&#39;imagerie vivent dans des conditions qui r&eacute;v&egrave;lent l&#39;autophagie basale (avec traitement BafA1), ou &agrave; l&#39;inactivation de MTOR (ce qui se traduirait par induction de l&#39;autophagie). Side-by-side comparaison a montr&eacute; que sous l&#39;autophagie basale et induit, 100 % des autophagosomes apparue &agrave; proximit&eacute; de brins de ER. Dans les mesures effectu&eacute;es en parall&egrave;le, 40 % &eacute;taient &agrave; proximit&eacute; de mitochondries dans les deux conditions. Nous avons conclu que, dans les cellules HEK 293, autophagie basale est m&eacute;caniquement similaire &agrave; celle induite par l&#39;inactivation de MTOR dans tous les aspects examin&eacute;s.

Caract&eacute;ristiques et exigences de l&#39;autophagie basale dans les cellules HEK 293.

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

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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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