immuno-fluorescent-labeling-microtubules-centrosomal-proteins-ex-vivo

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in <em>Ex Vivo</em> Intestinal Tissue and 3D <em>In Vitro</em> Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

University of East Anglia

The replication of many viruses is associated with specific intracellular compartments called virus factories or virioplasm. These are thought to provide a physical scaffold to concentrate viral components and thereby increase the efficiency of replication. The formation of virus replication sites often results in rearrangement of cellular membranes and reorganization of the cytoskeleton. Similar rearrangements are seen in cells in response to protein aggregation, where aggresomes and autophagosomes are produced to facilitate protein degradation. Here I review the evidence that some viruses induce aggresomes and autophagosomes to generate sites of replication.

Aggresomes and autophagy generate sites for virus replication.

Virus replication and virus assembly often occur in virus inclusions or virus factories that form at pericentriolar sites close to the microtubule organizing center or in specialized nuclear domains called ND10/PML bodies. Similar inclusions called aggresomes form in response to protein aggregation. Protein aggregates are toxic to cells and are transported along microtubules to aggresomes for immobilization and subsequent degradation by proteasomes and/or autophagy. The similarity between aggresomes and virus inclusions raises the possibility that viruses use aggresome pathways to concentrate cellular and viral proteins to facilitate replication and assembly. Alternatively, aggresomes may be part of an innate cellular response that recognizes virus components as foreign or misfolded and targets them for storage and degradation. Insights into the possible roles played by aggresomes during virus assembly are emerging from an understanding of how virus inclusions form and how viral proteins are targeted to them.

Aggresomes and pericentriolar sites of virus assembly: cellular defense or viral design?

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.

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&#xFF08;&#x4F8B;&#x5982;&#xFF0C;autophagosomes &#x6216; autolysosomes&#xFF09; &#x7684;&#x6D4B;&#x91CF;&#x7ED3;&#x679C;&#x4E4B;&#x95F4;&#x7684;&#x5DEE;&#x5F02;&#x4E0E;&#x90A3;&#x4E9B;&#x81EA;&#x566C;&#x8FC7;&#x7A0B;&#x7684;&#x4EFB;&#x4F55;&#x9636;&#x6BB5;&#xFF0C;&#x68C0;&#x6D4B;&#x6D41;&#x91CF;&#x901A;&#x8FC7;&#x81EA;&#x566C;&#x9014;&#x5F84; &#xFF08;&#x5373;&#xFF0C;&#x5B8C;&#x6574;&#x7684;&#x8FDB;&#x7A0B;&#xFF09; &#xFF1B;&#x56E0;&#x6B64;&#xFF0C;macroautophagy autophagosome &#x79EF;&#x7D2F;&#x7684;&#x7ED3;&#x679C;&#x9700;&#x8981;&#x52A0;&#x4EE5;&#x533A;&#x5206;&#x4ECE;&#x81EA;&#x566C;&#x6027;&#x6D3B;&#x52A8;&#x589E;&#x52A0;&#xFF0C;&#x4F1A;&#x5BFC;&#x81F4;&#x7684;&#x523A;&#x6FC0;&#x4E2D;&#x7684;&#x5757;&#x5B9A;&#x4E49;&#x4E3A;&#x589E;&#x52A0;&#x81EA;&#x566C;&#x8BF1;&#x5BFC;&#x52A0;&#x4E0A;&#x589E;&#x52A0;&#x7684;&#x4EA4;&#x4ED8;&#x548C;&#x6EB6;&#x9176;&#x4F53; &#xFF08;&#x5728;&#x5927;&#x591A;&#x6570;&#x9AD8;&#x7B49;&#x771F;&#x6838;&#x751F;&#x7269;&#x548C;&#x76D8;&#x57FA;&#x7F51;&#x67C4;&#x7B49;&#x4E00;&#x4E9B;&#x751F;&#x7269;&#x4F53;&#xFF09; &#x6216; &#xFF08;&#x5728;&#x690D;&#x7269;&#x548C;&#x771F;&#x83CC;&#xFF09; &#x6CE1;&#x5185;&#x7684;&#x964D;&#x89E3;&#x3002;&#x6362;&#x53E5;&#x8BDD;&#x8BF4;&#xFF0C;&#x5B83;&#x662F;&#x5C24;&#x5176;&#x91CD;&#x8981;&#x7684;&#x662F;&#x65B0;&#x7684;&#x9886;&#x57DF;&#x7684;&#x8C03;&#x67E5;&#x4EBA;&#x5458;&#x4E86;&#x89E3;&#x66F4;&#x591A; autophagosomes &#x7684;&#x5916;&#x89C2;&#x4E0D;&#x4E00;&#x5B9A;&#x7B49;&#x540C;&#x66F4;&#x591A;&#x81EA;&#x566C;&#x3002;&#x4E8B;&#x5B9E;&#x4E0A;&#xFF0C;&#x5728;&#x8BB8;&#x591A;&#x60C5;&#x51B5;&#x4E0B;&#xFF0C;autophagosomes &#x79EF;&#x7D2F;&#x7531;&#x4E8E;&#x8D29;&#x8FD0;&#x5230;&#x6EB6;&#x9176;&#x4F53;&#x4E0D;&#x968F;&#x4E4B;&#x6539;&#x53D8;&#x7684;&#x751F;&#x7269;&#x53D1;&#x751F; autophagosome&#xFF0C;&#x4E00;&#x4E2A;&#x5757;&#x800C;&#x589E;&#x52A0; autolysosomes &#x53EF;&#x964D;&#x89E3;&#x6D3B;&#x52A8;&#x4E2D;&#x53CD;&#x6620;&#x51CF;&#x5C11;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x63D0;&#x51FA;&#x4E00;&#x5957;&#x51C6;&#x5219;&#x7684;&#x9009;&#x62E9;&#x548C;&#x4F7F;&#x7528;&#x65B9;&#x6CD5;&#x7684;&#x89E3;&#x91CA;&#x7531;&#x8C03;&#x67E5;&#x4EBA;&#x5458;&#xFF0C;&#x4ED6;&#x4EEC;&#x7684;&#x76EE;&#x7684;&#x662F;&#x68C0;&#x67E5; 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In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy.

En 2008, nous avons publi&eacute; le premier ensemble de lignes directrices pour la normalisation des recherches dans l&#39;autophagie. Depuis lors, les recherches sur ce sujet a continu&eacute; d&#39;acc&eacute;l&eacute;rer, et beaucoup de scientifiques nouveaux entr&eacute;s dans le champ. Notre base de connaissances et les nouvelles technologies ont &eacute;galement &eacute;tendu. En cons&eacute;quence, il est important de mettre &agrave; jour ces lignes directrices pour la surveillance de l&#39;autophagie dans diff&eacute;rents organismes. Diff&eacute;rents examens ont d&eacute;crit les divers essais qui ont &eacute;t&eacute; utilis&eacute;s &agrave; cet effet. N&eacute;anmoins, il continue d&#39;y avoir confusion au sujet des m&eacute;thodes acceptables pour mesurer l&#39;autophagie, surtout chez les eucaryotes multicellulaires. Un point essentiel qui doit &ecirc;tre soulign&eacute;, c&#39;est qu&#39;il y a une diff&eacute;rence entre les diff&eacute;rentes mesures qui surveillent les chiffres ou le volume des &eacute;l&eacute;ments autophagiques (autophagosomes ou autolysosomes) &agrave; tout stade du processus autophagique vs ceux ce flux de mesure par la voie de l&#39;autophagie (c.-&agrave;-d. le processus complet) ; ainsi, un bloc en macroautophagy, ce qui entra&icirc;ne l&#39;accumulation autophagosome doit &ecirc;tre diff&eacute;renci&eacute;s des stimuli qui r&eacute;sultent dans l&#39;augmentation de l&#39;activit&eacute; autophagique, d&eacute;finie comme augmentation autophagie induction coupl&eacute;e avec livraison accrue &agrave; et d&eacute;gradation dans les lysosomes (dans la plupart des eucaryotes sup&eacute;rieurs et certains protistes comme Dictyostelium) ou la vacuole (chez les plantes et les champignons). En d&#39;autres termes, il est particuli&egrave;rement important que les chercheurs &agrave; nouveau dans le domaine comprennent que l&#39;apparence des autophagosomes plus ne correspond pas n&eacute;cessairement avec autophagie plus. En effet, dans de nombreux cas, autophagosomes s&#39;accumulent en raison d&#39;un bloc dans le trafic vers les lysosomes sans changement concomitant autophagosome biogen&egrave;se, alors qu&#39;une augmentation d&#39;autolysosomes peut refl&eacute;ter une r&eacute;duction dans l&#39;activit&eacute; de d&eacute;gradation. Nous pr&eacute;sentons ici une s&eacute;rie de lignes directrices pour la s&eacute;lection et l&#39;interpr&eacute;tation des m&eacute;thodes pour l&#39;utilisation par les enqu&ecirc;teurs, qui visent &agrave; examiner les macroautophagy et les processus connexes, ainsi que pour les examinateurs qui doivent fournir des critiques r&eacute;alistes et raisonnables des papiers qui mettent l&#39;accent sur ces processus. Ces lignes directrices ne visent pas &agrave; &ecirc;tre un ensemble de formule de r&egrave;gles, parce que les dosages appropri&eacute;s d&eacute;pendent en partie de la question pos&eacute;e et le syst&egrave;me utilis&eacute;. En outre, nous soulignons qu&#39;aucun dosage individuel n&#39;est garanti d&#39;&ecirc;tre le plus appropri&eacute; dans chaque situation, et nous recommandons fortement l&#39;utilisation de multiples essais pour surveiller l&#39;autophagie. Dans ces lignes directrices, nous consid&eacute;rons ces diverses m&eacute;thodes d&#39;&eacute;valuation d&#39;autophagie et quels renseignements peuvent, ou ne peut &ecirc;tre obtenu d&#39;eux. Enfin, en discutant les avantages et les limites des analyses particuli&egrave;res autophagie, nous esp&eacute;rons encourager l&#39;innovation technique dans le domaine.

Lignes directrices pour l&#39;utilisation et l&#39;interpr&eacute;tation d&#39;essais pour la surveillance d&#39;autophagie.

Im Jahr 2008 haben wir den ersten Satz von Richtlinien f&uuml;r die Standardisierung der Forschung in Autophagy ver&ouml;ffentlicht. Seitdem Forschung zu diesem Thema hat sich weiter beschleunigen, und viele neue Wissenschaftler haben das Spielfeld betreten. Unsere Wissensdatenbank und relevante neue Technologien haben auch erweitert. Dementsprechend ist es wichtig, diese Leitlinien f&uuml;r die &Uuml;berwachung Autophagy in verschiedenen Organismen zu aktualisieren. Verschiedene Meinungen haben die Proben beschrieben, die f&uuml;r diesen Zweck verwendet wurden. Dennoch weiterhin besteht Verwirrung in Bezug auf akzeptierte, Autophagy, vor allem bei mehrzelligen Eukaryoten zu messen. Ein wesentlicher Punkt, der betont werden muss ist, dass es ein Unterschied zwischen Messungen, die &Uuml;berwachung der Zahlen oder Volume autophagic Elemente (z. B. Autophagosomes oder Autolysosomes) in jedem Stadium des Prozesses gegen die autophagic dieser Ma&szlig;nahme-Fluss durch den Autophagy Pathway (d. h. der vollst&auml;ndige Proze&szlig;); so ein Block in Macroautophagy, Ergebnisse in Autophagosomen Anh&auml;ufung von Reize unterschieden werden, die erh&ouml;hte autophagic Aktivit&auml;t, f&uuml;hren die definiert als erh&ouml;hte Autophagy Induktion gepaart mit erh&ouml;hter Lieferung an und Verschlechterung in Lysosomen (in den meisten h&ouml;heren Eukaryotes und einigen Einzellern wie Dictyostelium) oder die Vakuole (in Pflanzen und Pilzen). Mit anderen Worten, ist es besonders wichtig, dass die Ermittler in das Feld neue verstehen, dass die Darstellung des mehr Autophagosomes nicht unbedingt mit mehr Autophagy gleichzusetzen ist. In der Tat sammeln in vielen F&auml;llen Autophagosomes wegen einen Block in den Handel zu Lysosomen ohne eine damit einhergehende &Auml;nderung in Autophagosomen Biogenese, w&auml;hrend eine Zunahme der Autolysosomes eine Verringerung in Degradations-Aktivit&auml;t widerspiegeln kann. Hier pr&auml;sentieren wir eine Reihe von Leitlinien f&uuml;r die Auswahl und Interpretation der Methoden f&uuml;r die Verwendung von Ermittlern, die Macroautophagy und die damit verbundenen Prozesse untersuchen wollen, sowie f&uuml;r Pr&uuml;fer, die realistische und vern&uuml;nftige Kritiken von Papier bereitstellen, die auf diese Prozesse gerichtet werden m&uuml;ssen. Diese Richtlinien sollen kein formelhaften Satz von Regeln, weil die entsprechenden Assays teilweise auf die Frage und das System verwendet wird. Dar&uuml;ber hinaus betonen wir, dass keine einzelne Assay ist garantiert das passende Produkt in jeder Situation, und wir empfehlen die Verwendung von mehreren Proben Autophagy &uuml;berwachen. In diesen Leitlinien halten wir diese verschiedenen Methoden zur Bewertung Autophagy und welche Informationen kann, oder nicht, von ihnen abgerufen werden. Abschlie&szlig;end diskutieren die Vorz&uuml;ge und Grenzen von bestimmten Autophagy Assays, hoffen wir auf technische Innovationen im Bereich zu f&ouml;rdern.

Richtlinien f&uuml;r die Verwendung und Interpretation von Assays zur &Uuml;berwachung Autophagy in Verbindung.

Nel 2008 abbiamo pubblicato la prima serie di linee guida per la standardizzazione della ricerca in autophagy. Da allora, ricerca su questo argomento ha continuato ad accelerare, e molti nuovi scienziati hanno entrato in campo. Hanno anche ampliato la nostra knowledge base e le nuove tecnologie pertinenti. Di conseguenza, &egrave; importante aggiornare queste linee guida per il monitoraggio autophagy in diversi organismi. Varie recensioni hanno descritto la gamma di dosaggi che sono stati utilizzati per questo scopo. Tuttavia, ci continua a essere confusione per quanto riguarda i metodi accettabili per misurare autophagy, specialmente negli eucarioti pluricellulari. Un punto chiave che deve essere sottolineato &egrave; che c&#39;&egrave; una differenza tra le misure che monitorare il volume di autophagic elementi (ad esempio, autophagosomes o autolysosomes) o numeri in qualsiasi fase del processo autophagic vs quelli che misura flusso attraverso il pathway autophagy (cio&egrave;, il processo completo); cos&igrave;, un blocco in macroautophagy che risultati in accumulo di autophagosome deve essere differenziato da stimoli che si traducono in maggiore attivit&agrave; autophagic, definita come aumento autophagy induzione accoppiato con consegna aumentata e la degradazione nei lisosomi (nella maggior parte dei eucarioti superiori e alcuni protisti come Dictyostelium) o il vacuolo (in piante e funghi). In altre parole, &egrave; particolarmente importante che gli investigatori di nuovi al campo capiscano che l&#39;aspetto di pi&ugrave; autophagosomes non necessariamente identificare con pi&ugrave; autophagy. Infatti, in molti casi, autophagosomes si accumulano a causa di un blocco nel traffico ai lisosomi senza un cambiamento concomitante di autophagosome biogenesi, considerando che un aumento di autolysosomes pu&ograve; riflettere una riduzione nell&#39;attivit&agrave; degradanti. Qui, vi presentiamo una serie di linee guida per la selezione e l&#39;interpretazione dei metodi per l&#39;uso dagli investigatori che mirano a esaminare macroautophagy e dei processi correlati, nonch&eacute; per i revisori che hanno bisogno di fornire critiche realistiche e ragionevole delle carte che sono focalizzate su questi processi. Queste linee guida non sono destinate ad essere una serie stereotipata di regole, perch&eacute; i dosaggi appropriati dipendono in parte la domanda e il sistema in uso. Inoltre, sottolineiamo che nessun dosaggio individuale &egrave; garantito per essere quello pi&ugrave; appropriato in ogni situazione, e si consiglia vivamente l&#39;uso di dosaggi multipli di monitorare autophagy. In queste linee guida, consideriamo questi vari metodi di valutazione autophagy e quali informazioni possono, o non pu&ograve; essere ottenuto da loro. Infine, da discutere i meriti e i limiti delle analisi particolare autophagy, speriamo di incoraggiare l&#39;innovazione tecnica nel campo.

Linee guida per l&#39;uso e l&#39;interpretazione delle analisi per il monitoraggio autophagy.

2008 &#x5E74;&#x306B;&#x6700;&#x521D;&#x306E;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x306E;&#x7814;&#x7A76;&#x6A19;&#x6E96;&#x5316;&#x306E;&#x305F;&#x3081;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x306E;&#x30BB;&#x30C3;&#x30C8;&#x3092;&#x516C;&#x958B;&#x3057;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E;&#x5F8C;&#x3001;&#x3053;&#x306E;&#x30C8;&#x30D4;&#x30C3;&#x30AF;&#x306B;&#x95A2;&#x3059;&#x308B;&#x7814;&#x7A76;&#x3092;&#x52A0;&#x901F;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x7D9A;&#x3051;&#x3066;&#x3044;&#x308B;&#x3001;&#x591A;&#x304F;&#x306E;&#x65B0;&#x3057;&#x3044;&#x79D1;&#x5B66;&#x8005;&#x53C2;&#x5165;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x79C1;&#x9054;&#x306E;&#x77E5;&#x8B58;&#x30D9;&#x30FC;&#x30B9;&#x3068;&#x95A2;&#x9023;&#x3059;&#x308B;&#x65B0;&#x3057;&#x3044;&#x30C6;&#x30AF;&#x30CE;&#x30ED;&#x30B8;&#x3082;&#x62E1;&#x5927;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x7570;&#x306A;&#x308B;&#x751F;&#x7269;&#x306B;&#x304A;&#x3051;&#x308B;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3053;&#x308C;&#x3089;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x3092;&#x66F4;&#x65B0;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;&#x69D8;&#x3005; &#x306A;&#x30EC;&#x30D3;&#x30E5;&#x30FC;&#x306F;&#x3053;&#x306E;&#x76EE;&#x7684;&#x306E;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E;&#x7BC4;&#x56F2;&#x306B;&#x3064;&#x3044;&#x3066;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x305D;&#x308C;&#x306B;&#x3082;&#x304B;&#x304B;&#x308F;&#x3089;&#x305A;&#x3001;&#x7279;&#x306B;&#x591A;&#x7D30;&#x80DE;&#x771F;&#x6838;&#x751F;&#x7269;&#x306B;&#x304A;&#x3051;&#x308B;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3092;&#x6E2C;&#x5B9A;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x53D7;&#x3051;&#x5165;&#x308C;&#x65B9;&#x6CD5;&#x306B;&#x95A2;&#x3059;&#x308B;&#x6DF7;&#x4E71;&#x7D9A;&#x3051;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x91CD;&#x8981;&#x306A;&#x30DD;&#x30A4;&#x30F3;&#x30C8;&#x3092;&#x5F37;&#x8ABF;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x756A;&#x53F7;&#x307E;&#x305F;&#x306F;&#x30DC;&#x30EA;&#x30E5;&#x30FC;&#x30E0; autophagic &#x8981;&#x7D20; &#xFF08;&#x4F8B;&#x3048;&#x3070;&#x3001;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x307E;&#x305F;&#x306F; autolysosomes) &#x306E;&#x76E3;&#x8996;&#x6E2C;&#x5B9A;&#x306E;&#x9055;&#x3044;&#x3082;&#x306E;&#x5BFE; autophagic &#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x3069;&#x306E;&#x6BB5;&#x968E;&#x3067;&#x3082;&#x305D;&#x306E;&#x30E1;&#x30B8;&#x30E3;&#x30FC; &#x30D5;&#x30E9;&#x30C3;&#x30AF;&#x30B9; &#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x7D4C;&#x8DEF; (&#x3059;&#x306A;&#x308F;&#x3061;&#x3001;&#x5B8C;&#x4E86;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;); &#x3092;&#x4ECB;&#x3057;&#x3066;&#x3067;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x84C4;&#x7A4D;&#x306E;&#x7D50;&#x679C;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308B;&#x5897;&#x52A0;&#x306E; autophagic &#x6D3B;&#x52D5;&#x306E;&#x7D50;&#x679C;&#x523A;&#x6FC0;&#x304B;&#x3089;&#x533A;&#x5225;&#x3055;&#x308C;&#x308B;&#x305F;&#x3081;&#x306B;&#x3067;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x3092;&#x5B9A;&#x7FA9;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x5897;&#x52A0;&#x306E;&#x914D;&#x4FE1;&#x3068;&#x52A3;&#x5316;&#x30EA;&#x30BD;&#x30BD;&#x30FC;&#x30E0; &#xFF08;&#x306E;&#x307B;&#x3068;&#x3093;&#x3069;&#x9AD8;&#x7B49;&#x771F;&#x6838;&#x751F;&#x7269;&#x3068;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x539F;&#x751F;&#x751F;&#x7269;&#x7D30;&#x80DE;&#x6027;&#x7C98;&#x83CC;&#x306A;&#x3069;&#xFF09; &#x307E;&#x305F;&#x306F; &#xFF08;&#x306E;&#x690D;&#x7269;&#x3068;&#x83CC;&#x985E;&#xFF09; &#x6DB2;&#x80DE;&#x5185;&#x7D50;&#x5408;&#x5897;&#x52A0;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x8A98;&#x5C0E;&#x3068;&#x3057;&#x3066;&#x3002;&#x4ED6;&#x306E;&#x8A00;&#x8449;&#x3067;&#x306F;&#x3001;&#x635C;&#x67FB;&#x5B98;&#x306F;&#x30D5;&#x30A3;&#x30FC;&#x30EB;&#x30C9;&#x306B;&#x65B0;&#x3057;&#x3044;&#x8A73;&#x7D30;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x306E;&#x5916;&#x89B3;&#x306F;&#x3001;&#x5FC5;&#x305A;&#x3057;&#x3082;&#x3088;&#x308A;&#x591A;&#x304F;&#x306E;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3068;&#x3092;&#x4E00;&#x81F4;&#x3057;&#x306A;&#x3044;&#x3092;&#x7406;&#x89E3;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x7279;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;&#x5B9F;&#x969B;&#x306B;&#x306F;&#x3001;autolysosomes &#x306E;&#x5897;&#x52A0;&#x5206;&#x89E3;&#x6D3B;&#x6027;&#x306E;&#x6E1B;&#x5C11;&#x3092;&#x53CD;&#x6620;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x306B;&#x5BFE;&#x3057;&#x30EA;&#x30BD;&#x30BD;&#x30FC;&#x30E0;&#x4F75;&#x7528;&#x5909;&#x5316;&#x306A;&#x3057;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x5F62;&#x6210;&#x306B;&#x304A;&#x3051;&#x308B;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x3067;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x306E;&#x305F;&#x3081;&#x591A;&#x304F;&#x306E;&#x5834;&#x5408;&#x304C;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B4;&#x30BD;&#x30FC;&#x30E0;&#x84C4;&#x7A4D;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x4E00;&#x9023;&#x306E;&#x9078;&#x629E;&#x306E;&#x305F;&#x3081;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x3068;&#x5229;&#x7528;&#x65B9;&#x6CD5;&#x306E;&#x89E3;&#x91C8;&#x3068;&#x95A2;&#x9023;&#x3059;&#x308B;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3092;&#x691C;&#x8A0E;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x76EE;&#x6307;&#x3057;&#x3066;&#x8ABF;&#x67FB;&#x5B98;&#x306B;&#x3088;&#x3063;&#x3066;&#x3060;&#x3051;&#x3067;&#x306A;&#x304F;&#x3001;&#x6821;&#x95B2;&#x8005;&#x304C;&#x3053;&#x308C;&#x3089;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306B;&#x7126;&#x70B9;&#x3092;&#x5F53;&#x3066;&#x3066;&#x3044;&#x308B;&#x66F8;&#x985E;&#x306E;&#x73FE;&#x5B9F;&#x7684;&#x304B;&#x3064;&#x5408;&#x7406;&#x7684;&#x306A;&#x6279;&#x5224;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308B;&#x305F;&#x3081;&#x672C;.&#x9069;&#x5207;&#x306A;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E;&#x4E00;&#x90E8;&#x3001;&#x8CEA;&#x554F;&#x3068;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3044;&#x308B;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x308B;&#x305F;&#x3081;&#x306B;&#x5B9A;&#x578B;&#x7684;&#x306A;&#x4E00;&#x9023;&#x306E;&#x30EB;&#x30FC;&#x30EB;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x306F;&#x610F;&#x5473;&#x3055;&#x308C;&#x307E;&#x305B;&#x3093;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x3059;&#x3079;&#x3066;&#x306E;&#x72B6;&#x6CC1;&#x3067;&#x6700;&#x3082;&#x9069;&#x5207;&#x306A; 1 &#x3064;&#x306B;&#x500B;&#x3005; &#x306E;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306F;&#x4FDD;&#x8A3C;&#x3055;&#x308C;&#x307E;&#x305B;&#x3093;&#x304A;&#x3088;&#x3073;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x8907;&#x6570;&#x306E;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E;&#x4F7F;&#x7528;&#x3092;&#x304A;&#x52E7;&#x3081;&#x3092;&#x5F37;&#x8ABF;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x3067;&#x306F;&#x3001;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3068;&#x3069;&#x306E;&#x3088;&#x3046;&#x306A;&#x60C5;&#x5831;&#x3092;&#x3067;&#x304D;&#x308B;&#x3001;&#x8A55;&#x4FA1;&#x306E;&#x3053;&#x308C;&#x3089;&#x306E;&#x3055;&#x307E;&#x3056;&#x307E;&#x306A;&#x65B9;&#x6CD5;&#x3092;&#x691C;&#x8A0E;&#x3057;&#x3066;&#x304F;&#x3060;&#x3055;&#x3044;&#x307E;&#x305F;&#x306F;&#x305D;&#x308C;&#x3089;&#x304B;&#x3089;&#x53D6;&#x5F97;&#x3059;&#x308B;&#x3053;&#x3068;&#x306F;&#x3067;&#x304D;&#x307E;&#x305B;&#x3093;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x30E1;&#x30EA;&#x30C3;&#x30C8;&#x3068;&#x7279;&#x5B9A;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x306E;&#x8A66;&#x91D1;&#x306E;&#x9650;&#x754C;&#x3092;&#x8AD6;&#x8B70;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x30D5;&#x30A3;&#x30FC;&#x30EB;&#x30C9;&#x3067;&#x306E;&#x6280;&#x8853;&#x9769;&#x65B0;&#x3092;&#x5968;&#x52B1;&#x3059;&#x308B;&#x9858;&#x3063;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x4F7F;&#x7528;&#x3068;&#x89E3;&#x91C8;&#x30AA;&#x30FC;&#x30C8;&#x30D5;&#x30A1;&#x30B8;&#x30FC;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x8A66;&#x91D1;&#x306E;&#x305F;&#x3081;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x3002;

2008 &#xB144; &#xC6B0;&#xB9AC; &#xCD9C;&#xD310; &#xC5F0;&#xAD6C; autophagy&#xC758; &#xD45C;&#xC900;&#xD654;&#xC5D0; &#xB300; &#xD55C; &#xC9C0;&#xCE68;&#xC758; &#xCCAB; &#xC138;&#xD2B8;. &#xADF8; &#xC774;&#xD6C4;&#xB85C;, &#xAC00;&#xC18D;,&#xC774; &#xC8FC;&#xC81C;&#xC5D0; &#xB300; &#xD55C; &#xC5F0;&#xAD6C;&#xB294; &#xACC4;&#xC18D; &#xD558; &#xACE0; &#xB9CE;&#xC740; &#xC0C8;&#xB85C;&#xC6B4; &#xACFC;&#xD559;&#xC790; &#xD544;&#xB4DC;&#xB97C; &#xC785;&#xB825; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xAE30;&#xC220; &#xC790;&#xB8CC; &#xBC0F; &#xAD00;&#xB828; &#xC2E0;&#xAE30;&#xC220;&#xB3C4; &#xD655;&#xB300; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xB530;&#xB77C;, &#xADF8;&#xAC83;&#xC740; &#xB2E4;&#xB978; &#xC720;&#xAE30; &#xCCB4;&#xC5D0;&#xC11C; autophagy &#xBAA8;&#xB2C8;&#xD130;&#xB9C1;&#xC5D0; &#xB300; &#xD55C;&#xC774; &#xC9C0;&#xCE68;&#xC744; &#xC5C5;&#xB370;&#xC774;&#xD2B8; &#xD558;&#xB294; &#xAC83;&#xC774; &#xC911;&#xC694;&#xC785;&#xB2C8;&#xB2E4;. &#xB2E4;&#xC591; &#xD55C; &#xB9AC;&#xBDF0;&#xB294;&#xC774; &#xBAA9;&#xC801;&#xC744; &#xC704;&#xD574; &#xC0AC;&#xC6A9; &#xB41C; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC758; &#xBC94;&#xC704;&#xB97C; &#xC124;&#xBA85; &#xD588;&#xB2E4;. &#xADF8;&#xB7FC;&#xC5D0;&#xB3C4; &#xBD88;&#xAD6C; &#xD558; &#xACE0;, &#xAC70;&#xAE30;&#xC5D0; &#xD5C8;&#xC6A9; &#xD2B9;&#xD788; &#xB2E4;&#xC138;&#xD3EC; &#xC9C4;&#xD575;&#xC0DD;&#xBB3C;&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; autophagy &#xCE21;&#xC815; &#xBC29;&#xBC95;&#xC5D0; &#xAD00;&#xD55C; &#xD63C;&#xB780; &#xB418; &#xACE0; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xAC15;&#xC870; &#xD558;&#xB294; &#xD575;&#xC2EC; &#xD3EC;&#xC778;&#xD2B8;&#xAC00;&#xC785;&#xB2C8;&#xB2E4; &#xC22B;&#xC790; &#xB610;&#xB294; autophagic &#xC694;&#xC18C; (&#xC608;: autophagosomes &#xB610;&#xB294; autolysosomes)&#xC758; &#xBCFC;&#xB968;&#xC744; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xD558;&#xB294; &#xCE21;&#xC815;&#xC758; &#xCC28;&#xC774; &#xADF8; &#xB300; autophagic &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC758; &#xBAA8;&#xB4E0; &#xB2E8;&#xACC4;&#xC5D0;&#xC11C; autophagy &#xD1B5;&#xB85C; (&#xC989;, &#xC804;&#xCCB4; &#xD504;&#xB85C;&#xC138;&#xC2A4;);&#xB97C; &#xD1B5;&#xD574; &#xADF8; &#xCE21;&#xC815; &#xC720;&#xB7C9; &#xB530;&#xB77C;&#xC11C;, &#xACB0;&#xACFC;: autophagosome &#xCD95;&#xC801; &#xC99D;&#xAC00; autophagic &#xD65C;&#xB3D9;&#xC5D0;&#xC11C; &#xBC1C;&#xC0DD; &#xD558;&#xB294; &#xC790;&#xADF9;&#xC5D0;&#xC11C; &#xCC28;&#xBCC4;&#xD654; &#xD558;&#xB294; macroautophagy&#xC5D0;&#xC11C; &#xBE14;&#xB85D; &#xC99D;&#xAC00; autophagy &#xC720;&#xB3C4;, &#xC99D;&#xAC00; &#xC804;&#xB2EC;&#xACFC; &#xB9AC;&#xC18C;&#xC880; (&#xB300;&#xBD80;&#xBD84;&#xC758; &#xB192;&#xC740; &#xC9C4;&#xD575;&#xC0DD;&#xBB3C;&#xC5D0; Dictyostelium &#xAC19;&#xC740; &#xC77C;&#xBD80; protists) &#xB610;&#xB294; (&#xC2DD;&#xBB3C;&#xACFC; &#xACF0; &#xD321;&#xC774;)&#xC5D0; &#xACF5;&#xD3EC; &#xB0B4; &#xC800;&#xD558;&#xC640; &#xACB0;&#xD569;&#xC73C;&#xB85C; &#xC815;&#xC758; &#xB429;&#xB2C8;&#xB2E4;. &#xB2E4;&#xB978; &#xB9D0;&#xB85C; &#xD558;&#xBA74;, &#xADF8;&#xAC83;&#xC740; &#xD2B9;&#xD788; &#xC911;&#xC694; &#xD55C; &#xC218;&#xC0AC; &#xBD84;&#xC57C;&#xC5D0; &#xC0C8;&#xB85C;&#xC6B4; &#xB354; &#xB9CE;&#xC740; autophagosomes&#xC758; &#xC678;&#xAD00;&#xC744; &#xBC18;&#xB4DC;&#xC2DC; &#xB354; autophagy&#xC640; &#xB3D9;&#xC77C;&#xC2DC; &#xD558;&#xC9C0; &#xC54A;&#xB294;&#xB2E4; &#xC774;&#xD574;. &#xC0AC;&#xC2E4;, &#xB300;&#xBD80;&#xBD84;&#xC758; &#xACBD;&#xC6B0;&#xC5D0;&#xC11C; autophagosomes &#xCD95;&#xC801; autophagosome &#xC18D;&#xC5D0; &#xC218; &#xBC18;&#xD558;&#xB294; &#xBCC0;&#xD654; &#xC5C6;&#xC774; &#xB9AC;&#xC18C;&#xC880;&#xC744; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC5D0; &#xBE14;&#xB85D; &#xB54C;&#xBB38;&#xC5D0; &#xBC18;&#xBA74;&#xC5D0; autolysosomes &#xC99D;&#xAC00; degradative &#xD65C;&#xB3D9;&#xC758; &#xAC10;&#xC18C;&#xB97C; &#xBC18;&#xC601; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; &#xD604;&#xC7AC; &#xC120;&#xD0DD; &#xC601;&#xC5ED;&#xC5D0; &#xB300; &#xD55C; &#xC9C0;&#xCE68;&#xC758; &#xC124;&#xC815; &#xBC0F; &#xC0AC;&#xC6A9; &#xBC29;&#xBC95;&#xC758; &#xD574;&#xC11D; &#xC774;&#xB7EC;&#xD55C; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC5D0; &#xC9D1;&#xC911; &#xB418;&#xB294; &#xB17C;&#xBB38;&#xC758; &#xD604;&#xC2E4;&#xC801;&#xC774; &#xACE0; &#xD569;&#xB9AC;&#xC801;&#xC778; &#xBE44;&#xD310;&#xC744; &#xC81C;&#xACF5; &#xD574;&#xC57C; &#xD558;&#xB294; &#xAC80;&#xD1A0;&#xC790;&#xC5D0; &#xB300; &#xD55C; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; macroautophagy &#xBC0F; &#xAD00;&#xB828;&#xB41C; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xB97C; &#xAC80;&#xC0AC; &#xD558;&#xB294; &#xAC83;&#xC744; &#xBAA9;&#xD45C;&#xB85C; &#xD558;&#xB294; &#xC870;&#xC0AC;&#xC5D0; &#xC758;&#xD574;. &#xC801;&#xC808; &#xD55C; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8; &#xBD80;&#xBD84;&#xC5D0;&#xC11C; &#xC694;&#xCCAD;&#xC744; &#xBC1B;&#xACE0; &#xC9C8;&#xBB38;&#xC744; &#xD558; &#xACE0; &#xC0AC;&#xC6A9; &#xC911;&#xC778; &#xC2DC;&#xC2A4;&#xD15C;&#xC5D0; &#xC758;&#xC874; &#xD558;&#xAE30; &#xB54C;&#xBB38;&#xC5D0; &#xC774;&#xB7EC;&#xD55C; &#xC9C0;&#xCE68; &#xADDC;&#xCE59;, formulaic &#xC9D1;&#xD569;&#xC73C;&#xB85C; &#xC758;&#xBBF8;&#xAC00; &#xC5C6;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC;&#xB294; &#xC544;&#xBB34; &#xAC1C;&#xBCC4; &#xBD84;&#xC11D; &#xACB0;&#xACFC;, &#xBAA8;&#xB4E0; &#xC0C1;&#xD669;&#xC5D0;&#xC11C; &#xAC00;&#xC7A5; &#xC801;&#xD569; &#xD55C; &#xBCF4;&#xC7A5; &#xD558; &#xACE0; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4; autophagy &#xBAA8;&#xB2C8;&#xD130;&#xB9C1;&#xD560; &#xC5EC;&#xB7EC; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC758; &#xC0AC;&#xC6A9;&#xC744; &#xAC15;&#xC870;. &#xC774;&#xB7EC;&#xD55C; &#xC9C0;&#xCE68;&#xC5D0; &#xC6B0;&#xB9AC; autophagy&#xC640; &#xC815;&#xBCF4; &#xC218; &#xC788;&#xB294; &#xD3C9;&#xAC00;&#xC758; &#xC774;&#xB7EC;&#xD55C; &#xB2E4;&#xC591; &#xD55C; &#xBC29;&#xBC95;&#xC744; &#xACE0;&#xB824; &#xADF8;&#xB4E4; &#xB85C;&#xBD80;&#xD130; &#xC5BB;&#xC744; &#xC218; &#xC5C6;&#xC2B5;&#xB2C8;&#xB2E4;, &#xB610;&#xB294;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, &#xC7A5;&#xC810; &#xBC0F; &#xD2B9;&#xC815; autophagy &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC758; &#xD55C;&#xACC4;&#xB97C; &#xB17C;&#xC758; &#xD558; &#xC5EC; &#xD604;&#xC7A5;&#xC5D0;&#xC11C; &#xAE30;&#xC220; &#xD601;&#xC2E0;&#xC744; &#xC7A5;&#xB824; &#xD558;&#xACA0;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xC0AC;&#xC6A9; &#xBC0F; autophagy &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC758; &#xD574;&#xC11D;&#xC5D0; &#xB300; &#xD55C; &#xC9C0;&#xCE68;.

En 2008 public&oacute; el primer conjunto de pautas para estandarizar la investigaci&oacute;n en la autofagia. Desde entonces, ha continuado la investigaci&oacute;n sobre este tema acelerar, y muchos nuevos cient&iacute;ficos han entrado en el campo. Tambi&eacute;n han estado ampliando nuestra base de conocimientos y tecnolog&iacute;as relevantes. Por consiguiente, es importante actualizar estas directrices para la supervisi&oacute;n de autofagia en diferentes organismos. Varios comentarios han descrito la gama de ensayos que se han utilizado para este prop&oacute;sito. Sin embargo, contin&uacute;an confusi&oacute;n con respecto a los m&eacute;todos aceptables para medir la autofagia, especialmente en eucariotas pluricelulares. Un punto clave que debe destacarse es que hay una diferencia entre las medidas que monitorean los n&uacute;meros o volumen de autofagia elementos (por ejemplo, autophagosomes o autolysosomes) en cualquier etapa del proceso de autofagia vs los ese flujo medida a trav&eacute;s de la v&iacute;a de la autofagia (es decir, el proceso completo); as&iacute;, un bloque en macroautophagy que resulta en acumulaci&oacute;n de autophagosome debe ser distinguido de est&iacute;mulos que resultan en un aumento de la actividad autofagia, definido como inducci&oacute;n autofagia creciente juntada con mayor entrega y degradaci&oacute;n dentro, lisosomas (en la mayor&iacute;a de eucariotas superiores y algunos protistas como Dictyostelium) o la vacuola (en plantas y hongos). En otras palabras, es especialmente importante que los investigadores de nuevos al campo entiendan que la aparici&oacute;n de m&aacute;s autophagosomes no corresponde necesariamente con autofagia m&aacute;s. De hecho, en muchos casos, autophagosomes se acumulan debido a un bloque en el tr&aacute;fico a los lisosomas sin un cambio concomitante en la biog&eacute;nesis de autophagosome, mientras que un aumento en la autolysosomes puede reflejar una reducci&oacute;n en la actividad de degradaci&oacute;n. Aqu&iacute;, presentamos un conjunto de directrices para la selecci&oacute;n e interpretaci&oacute;n de los m&eacute;todos para su uso por los investigadores que tienen como objetivo examinar macroautophagy y procesos relacionados, as&iacute; como para los revisores que deban proporcionar cr&iacute;ticas realistas y razonables de papeles que se centran en estos procesos. Estas directrices no pretenden ser un formulista conjunto de reglas, porque los ensayos adecuados dependen en parte de la pregunta y el sistema utilizado. Adem&aacute;s, hacemos hincapi&eacute; en que ning&uacute;n ensayo individual est&aacute; garantizada para ser el m&aacute;s adecuado en cada situaci&oacute;n, y le recomendamos el uso de m&uacute;ltiples ensayos para monitorear la autofagia. En estas directrices, consideramos estos diversos m&eacute;todos de evaluaci&oacute;n de autofagia y qu&eacute; informaci&oacute;n puede, o no puede obtenerse de ellos. Finalmente, discutiendo los m&eacute;ritos y los l&iacute;mites de an&aacute;lisis particular de la autofagia, queremos fomentar la innovaci&oacute;n t&eacute;cnica en el campo.

Directrices para el uso e interpretaci&oacute;n de ensayos para el control de la autofagia.

Photoinduced electron transfer (PET)-based molecular probes have been successfully used for the intracellular imaging of the pH of acidic organelles. In this study, we describe the synthesis and characterization of a novel PET-based pH nanoprobe and its biological application for the signaling of acidic organelles in mammalian cells. A fluorescent ligand sensitive to pH via the PET mechanism that incorporates a thiolated moiety was synthesized and used to stabilize gold nanoparticles (2.4 ± 0.6 nm), yielding a PET-based nanoprobe. The PET nanoprobe was unambiguously characterized by transmission electron microscopy, proton nuclear magnetic resonance, Fourier transform infrared, ultraviolet-visible absorption, and steady-state/time-resolved fluorescence spectroscopies which confirmed the functionalization of the gold nanoparticles with the PET-based ligand. Following a classic PET behavior, the fluorescence emission of the PET-based nanoprobe was quenched in alkaline conditions and enhanced in an acidic environment. The PET-based nanoprobe was used for the intracellular imaging of acidic environments within Chinese hamster ovary cells by confocal laser scanning microscopy. The internalization of the nanoparticles by the cells was confirmed by confocal fluorescence images and also by recording the fluorescence emission spectra of the intracellular PET-based nanoprobe from within the cells. Co-localization experiments using a marker of acidic organelles, LysoTracker Red DND-99, and a marker of autophagosomes, GFP-LC3, confirm that the PET-based nanoprobe acts as marker of acidic organelles and autophagosomes within mammalian cells.

A photoinduced electron transfer-based nanoprobe as a marker of acidic organelles in mammalian cells.

Autophagy is a membrane trafficking pathway that results in the formation of autophagosomes which deliver portions of the cytosol to lysosomes for degradation. When autophagosomes engulf intracellular pathogens, the pathway is called 'xenophagy' because it leads to the removal of foreign material. Autophagy is activated during infection by Toll-like receptors that recognize pathogen-associated molecular patterns. This allows autophagy to kill micro-organisms and present pathogen components to the innate and acquired immune systems. The targeting of pathogens by autophagy is selective and involves a growing family of autophagy receptors that bind to the autophagosome membrane protein LC3 (light-chain 3)/Atg8 (autography-related protein 8). Ubiquitination of microbes identifies them as substrates for autophagy and they are delivered to autophagosomes by autophagy receptors that bind both ubiquitin and LC3/Atg8. Bacteria can also be detected before they enter the cytosol by autophagy receptors that scan the surface of membrane compartments for evidence of damage. The observation that some pathogens survive in cells suggests they can evade complete destruction by autophagy. For some bacteria this involves proteins that shield the surface of the bacteria from recognition by autophagy receptors. Other viruses and bacteria are resistant to degradation in lysosomes and use autophagosomes and/or lysosomes as sites for replication. Most of our current understanding of the role played by autophagy during microbial infection has come from studies of bacteria and viruses in tissue culture cell lines. Future work will focus on understanding how autophagy determines the outcome of infection 'in vivo', and how autophagy pathways can be exploited therapeutically.

Autophagy as a defence against intracellular pathogens.

Replication of positive-sense RNA viruses is associated with the rearrangement of cellular membranes. Previous work on the infection of tissue culture cell lines with the betacoronaviruses mouse hepatitis virus and severe acute respiratory syndrome coronavirus (SARS-CoV) showed that they generate double-membrane vesicles (DMVs) and convoluted membranes as part of a reticular membrane network. Here we describe a detailed study of the membrane rearrangements induced by the avian gammacoronavirus infectious bronchitis virus (IBV) in a mammalian cell line but also in primary avian cells and in epithelial cells of ex vivo tracheal organ cultures. In all cell types, structures novel to IBV infection were identified that we have termed zippered endoplasmic reticulum (ER) and spherules. Zippered ER lacked luminal space, suggesting zippering of ER cisternae, while spherules appeared as uniform invaginations of zippered ER. Electron tomography showed that IBV-induced spherules are tethered to the zippered ER and that there is a channel connecting the interior of the spherule with the cytoplasm, a feature thought to be necessary for sites of RNA synthesis but not seen previously for membrane rearrangements induced by coronaviruses. We also identified DMVs in IBV-infected cells that were observed as single individual DMVs or were connected to the ER via their outer membrane but not to the zippered ER. Interestingly, IBV-induced spherules strongly resemble confirmed sites of RNA synthesis for alphaviruses, nodaviruses, and bromoviruses, which may indicate similar strategies of IBV and these diverse viruses for the assembly of RNA replication complexes.

Infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a heterogeneous disorder of significant societal impact that is proposed to involve both host and environmentally derived aetiologies that may be autoimmune in nature. Immune-related symptoms of at least moderate severity persisting for prolonged periods of time are common in ME/CFS patients and B cell depletion therapy is of significant therapeutic benefit. The origin of these symptoms and whether it is infectious or inflammatory in nature is not clear, with seeking evidence of acute or chronic virus infections contributing to the induction of autoimmune processes in ME/CFS being an area of recent interest. This article provides a comprehensive review of the current evidence supporting an infectious aetiology for ME/CFS leading us to propose the novel concept that the intestinal microbiota and in particular members of the virome are a source of the "infectious" trigger of the disease. Such an approach has the potential to identify disease biomarkers and influence therapeutics, providing much-needed approaches in preventing and managing a disease desperately in need of confronting.

A Role for the Intestinal Microbiota and Virome in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)?

Differentiation of columnar epithelial cells involves a dramatic reorganization of the microtubules (MTs) and centrosomal components into an apico-basal array no longer anchored at the centrosome. Instead, the minus-ends of the MTs become anchored at apical non-centrosomal microtubule organizing centres (n-MTOCs). Formation of n-MTOCs is critical as they determine the spatial organization of MTs, which in turn influences cell shape and function. However, how they are formed is poorly understood. We have previously shown that the centrosomal anchoring protein ninein is released from the centrosome, moves in a microtubule-dependent manner and accumulates at n-MTOCs during epithelial differentiation. Here, we report using depletion and knockout (KO) approaches that ninein expression is essential for apico-basal array formation and epithelial elongation and that CLIP-170 is required for its redeployment to n-MTOCs. Functional inhibition also revealed that IQGAP1 and active Rac1 coordinate with CLIP-170 to facilitate microtubule plus-end cortical targeting and ninein redeployment. Intestinal tissue and in vitro organoids from the Clip1/Clip2 double KO mouse with deletions in the genes encoding CLIP-170 and CLIP-115, respectively, confirmed requirement of CLIP-170 for ninein recruitment to n-MTOCs, with possible compensation by other anchoring factors such as p150Glued and CAMSAP2 ensuring apico-basal microtubule formation despite loss of ninein at n-MTOCs.

Tom Wileman

Norwich Medical School

University of East Anglia

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

Tom Wileman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Norwich Medical School

University of East Anglia

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

Norwich Medical School