We thank the Northwestern University Mouse Histology and Phenotyping Laboratoryfor technical support and assistance, and Noboru Mizushima (University of Tokyo) for providing GFP-LC3 transgenic mice. A. R. and C. H. were supported by the startup funds from Northwestern University and the grant from National Institutes of Health (DK094980).

Tutte le procedure che coinvolgono gli animali sono stati eseguiti secondo le linee guida approvate dal Comitato Northwestern University istituzionale cura degli animali e Usa (IACUC). 1. modelli murini <ol><li> Utilizzare 8-12 settimane di età topi nella formazione esercizio. Per rilevare l&#39;autofagia esercitato indotta in vivo, utilizzare il topi transgenici GFP-LC3 (C57BL / 6 di fondo) per studi di imaging e C57BL / 6 topi per le analisi biochimiche. </li></ol> 2. L&#39;autofagia indotta da esercizio <ol><li> Messa a punto Treadmill - esercizio forzata <ol><li> Acclimatarsi e formare i topi in un 10 ° in salita tapis roulant aperto per 2 giorni. Il giorno 1, esercitare i topi per 5 min a 8 m / min, e il giorno 2, esercitare i topi per 5 min a 8 m / min seguito da un altro 5 min a 10 m / min 10. Incoraggiare i topi per eseguire delicatamente gomitata mano. </li><li> Il giorno 3 °, lasciare che i topi sottoposti a un singolo attacco di corsa per 90 minuti a 10 ° in salita tapis roulant, starting alla velocità di 10 m / min. Dopo 40 min, aumentare la velocità treadmill ad una velocità di 1 m / min ogni 10 min per un totale di 30 min, e quindi aumentare la velocità alla velocità di 1 m / min ogni 5 min fino a raggiungere 17 m / min, per un totale di 90 minuti di tempo di esercizio e 1.070 metri di corsa a distanza. </li><li> Impostare lo stimolo elettrico ad una gamma bassa intensità (1,2 Hz), e incoraggiare i topi a correre per evitare scosse piede ripetuto. </li><li> Per la raccolta dei tessuti, eutanasia i topi per inalazione isoflurano, seguita da dislocazione cervicale immediatamente dopo l&#39;esercizio. Altri metodi approvati eutanasia possono anche essere utilizzati, come CO2 o iniezione IP di altri anestetici. Il livello di autofagia non sembra essere influenzata da metodi di eutanasia. </li></ol></li><li> Messa a punto della ruota Running - esercizio volontario <ol><li> Posizionare una rotella del mouse-esecuzione (11,4 cm) all&#39;interno di una gabbia con un mouse a singolo alloggiati. </li><li> Verificare la capacità corrente con una contachilometri bicicletta.Impostare il parametro della ruota [distanza (in mm) per ogni rotazione completa della ruota] a 358 (11.4 * π). Misurare la distanza in esecuzione dopo 24 ore. </li><li> Lasciate il mouse correre volontariamente per 2 settimane con la ruota in esecuzione. Per la raccolta dei tessuti, sacrificare il mouse al mattino dopo il periodo di esecuzione. </li></ol></li></ol> 3. L&#39;autofagia Flux valutazione <ol><li> Per misurare il flusso autofagica in condizioni di riposo e di esercizio, trattare i topi con la clorochina inibitore autofagia per tre giorni e confrontarle con topi trattati con PBS. <ol><li> Sciogliere clorochina in PBS (5 mg / ml), e iniettare clorochina in topi per via intraperitoneale alla dose di 50 mg / kg / giorno per tre giorni consecutivi. Sacrificio topi e raccogliere i tessuti 3 ore dopo l&#39;ultima iniezione. </li><li> Per i topi esercitati, li pretrattare con clorochina per due giorni consecutivi alla dose di 50 mg / kg / giorno. Il terzo giorno, iniettare i topi con la stessa concentrazione di clorochina, e dopo90 min li fanno correre sul tapis roulant per altri 90 min. </li><li> Euthanize topi per inalazione isoflurano, seguita da dislocazione cervicale. Raccogliere tessuti immediatamente dopo l&#39;esecuzione, in modo che il tempo totale di incubazione della clorochina è la stessa di controllo (a riposo) topi (3 ore). NOTA: In alternativa, utilizzare una singola iniezione di clorochina per determinare il flusso autofagia nei tessuti di topo. </li></ol></li></ol> 4. Tessuto Raccolta e Fixation <ol><li> Prima della raccolta dei tessuti preparare tampone fosfato salino (PBS) e fresca 4% paraformaldeide (PFA, attenzione: dispositivi di protezione individuale richiesto) in PBS a un pH finale di 7,4. Conservare entrambe le soluzioni a 4 ° C. </li><li> Riempire 30 ml siringa con 15 - 20 ml di PFA 4% (per i topi GFP-LC3). Collegare la siringa con un ago G 20 del catetere. </li><li> Dopo l&#39;esercizio, eutanasia i topi per inalazione isoflurano, seguita da dislocazione cervicale. </li><li> Posto l&#39;animale su una superficie di lavoro pulita sul suo back. Tagliare e aprire la cavità toracica tagliando il diaframma per esporre il cuore. </li><li> Effettuare una piccola incisione sul fegato per lasciare il sangue uscire, e iniettare un totale di 15 ml 4% PFA lentamente nel ventricolo destro del cuore attraverso il catetere, fino al polmone e fegato girare completamente pallido. Profumato il mouse immediatamente dopo la sessione di esercizio, utilizzando una pompa a siringa con una portata costante di 90 ml / h. </li><li> Dopo la perfusione, rimuovere l&#39;ago e raccogliere i tessuti di interesse (ad esempio, del cervello 11 e muscolo scheletrico). Per muscolo scheletrico, sezionare vasto laterale. Brevemente, tirare la pelle della gamba all&#39;indietro per esporre i muscoli, localizzare il quadricipite femorale 12 e sezionare vasto laterale, che è il muscolo esterno collegato alla parte superiore dell&#39;osso femorale. </li><li> Per ulteriori fissazione e disidratazione, posizionare i tessuti di topi GFP-LC3 in 4% PFA per 24 ore a 4 ° C, e poi trasferirli al 15%saccarosio-PBS a 4 ° C durante la notte o fino a quando i tessuti sono stabiliti, e quindi al 30% di saccarosio-PBS a 4 ° C durante la notte o per più lunghi periodi. Conservare i campioni al buio per quanto possibile in quanto possono essere sensibili a luce intensa. </li><li> Per l&#39;analisi Western Blot scatto congelare i tessuti da C57BL / 6 topi direttamente in azoto liquido, e conservare a -80 ° C. </li></ol> 5. Analisi Imaging di GFP-LC3 Puncta <ol><li> processo Tissue <ol><li> Pre-trattare i tessuti (precedentemente fissate in PFA e conservati nel 30% di saccarosio) nel mezzo di inclusione come &quot;ottimali di taglio composto temperatura&quot; (OCT) per pochi minuti in una piccola capsula di Petri etichettati. </li><li> Trasferire il tessuto in una cryomold l&#39;etichetta che contiene abbastanza fresco di Office per coprire il tessuto. Orient superficie sezionamento verso il fondo della cryomold (sezione per il muscolo vasto laterale e sezione sagittale per la emi-cervello). Evitare la formazione di bolle. </li><li> freÈze campioni ponendo il cryomold per pochi minuti in un dispositivo di raffreddamento di schiuma coperto riempito con ghiaccio secco, fino PTOM diventa bianco. Avvolgere singoli campioni in fogli etichettati, chiuderli in un sacchetto di plastica, e conservarli a -80 ° C durante la notte o più a lungo. </li><li> Utilizzare un criostato per tagliare le sezioni di esempio con uno spessore di 10 micron, e montare le sezioni sulle slitte. Conservare i vetrini a -20 ° C, e sempre tenere i vetrini al riparo dalla luce, quando possibile. </li></ol></li><li> preparazione del vetrino <ol><li> Estrarre i vetrini dal congelatore e scongelare a temperatura ambiente. Coprire la sezione di tessuto con una goccia di mezzo di montaggio con DAPI. Collocare un vetrino di dimensioni adeguate in cima ed evitare la formazione di bolle. </li><li> Utilizzare smalto per sigillare i bordi del vetrino, consentire smalto si asciughi al buio a temperatura ambiente, e quindi memorizzare le diapositive in un contenitore a tenuta di luce a 4 ° C, o procedere con l&#39;imaging cattura di GFP-LC3 puncta. </li></ol></li><li> &lt;strong&gt; La quantificazione di GFP - LC3 puncta al microscopio a fluorescenza <ol><li> Eseguire epifluorescenza, catturando le immagini nella stessa condizione (esposizione e le impostazioni) per tutti i campioni. Per GFP, utilizzare una lunghezza d&#39;onda di emissione di 525 nm; per DAPI, usare 490 nm. Acquisire almeno dieci immagini al campione utilizzando un obiettivo 60X per vasto laterale e la regione della corteccia frontale del cervello. </li><li> Quantificare il numero di GFP-LC3 puncta in una zona di tessuto di 2.500 micron 2 in ogni immagine per l&#39;analisi statistica, accecato per gruppi sperimentali. Calcolare il numero medio di 10 immagini di ogni campione, utilizzando la funzione di misura automatica di &quot;conteggio degli oggetti&quot;. Impostazioni per il muscolo vasto laterale sono: Soglia L = 10, H = 200; 2X liscia; 1X pulito, e le impostazioni per la corteccia frontale del cervello sono: Soglia L = 11, H = 200; 2X liscia; 1X pulito. </li></ol></li></ol> 6. occidentale Analisi Blot su autofagia Marcatori <ol><li> &lt;strong&gt; processo Tissue <ol><li> Preparare il tampone di lisi: 50 mM Tris-HCl; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; inibitore della proteasi e inibitori della fosfatasi (appena aggiunto). </li><li> Rimuovere il tessuto di C57BL 6 topi / da -80 ° C di stoccaggio. Tagliare il tessuto muscolare in piccoli pezzi con una lama di rasoio prima il seguente processo. Aggiungere 800 ml (per emi-cervello) o 500 ml (per vasto laterale) di tampone di lisi freddo al campione. Omogeneizzare il campione a 4 ° C con un omogeneizzatore ad una velocità media. </li><li> Completamente lisare la miscela omogeneizzata per altri 30 minuti per 1 ora a 4 ° C con rotazione. </li><li> Centrifugare l&#39;omogeneizzato a 12.000 xg per 10 min a 4 ° C, scartare il pellet, e raccogliere il surnatante in una nuova provetta. </li></ol></li><li> analisi autofagia <ol><li> Determinare la concentrazione di proteine ​​di lisati di tessuto utilizzando il kit di test BCA Protein in base alle specifiche del costruttore. Normalizzare ogni campionealla stessa concentrazione. </li><li> Unire il campione con il tampone 2x Laemmli in un rapporto 1: 1, far bollire a 95 ° C per 5 - 10 minuti, e procedere con l&#39;analisi Western Blot su autofagia marcatori 13, 14, come ad esempio P62 (anticorpo anti-P62, diluizione 1: 500) e LC3 (anticorpo anti-LC3, diluizione 1: 500). Lessare i campioni immediatamente dopo l&#39;aggiunta del tampone, come più incubazione in ghiaccio può generare più bande degradate LC3. </li><li> Quantificare P62 e LC3 bande di analisi densitometria utilizzando ImageJ, e normalizzare il valore al corrispondente banda di actina. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Autofagia è un processo catabolico che fornisce energia e riduce citotossicità dalla degradazione lisosomiale di anticitoplasma o organelli danneggiati. Studiare l&#39;autofagia è importante capire la regolazione dell&#39;omeostasi cellulare e meccanismi di risposta allo stress. Nuovi modelli e metodologie stanno emergendo nel campo della ricerca 15, per studiare come alterata autofagia contribuisce a numerosi processi patologici 16, 17.</su...

Autofagia è un percorso di degradazione evolutivamente conservato, che viene indotta in risposta a varie condizioni di stress come la fame e ipossia 1, 2. Durante l&#39;autofagia, vescicole a doppia membrana, chiamato autofagosomi, incorporano componenti subcellulari inutili o danneggiati e trasportarli nei lisosomi per la degradazione 3. Autofagia basale è essenziale per la funzione cellulare e lo sviluppo dell&#39;organismo, e alterata autofagia basale è implicato in molti disturbi, tra cui neurodegenerazione, tumorigenesi e diabete di tipo 2 4, 5, 6. Il più noto autofagia induttore fisiologico è fame. Tuttavia, ha due limitazioni principali. In primo luogo, la fame richiede un lungo periodo di indurre l&#39;autofagia in modo efficace negli animali, ad esempio, 48 ore di restrizione alimentare nei topinella maggior parte degli organi. In secondo luogo, la fame induce a malapena autofagia cervello, a causa di un apporto di sostanze nutritive relativamente stabile nel cervello. In realtà, è anche difficile da rilevare induzione autofagia dagli induttori di piccole molecole, come molti farmaci non possono passare la barriera emato-encefalica. Così, per analizzare meglio la funzione di attivazione dell&#39;autofagia nella patogenesi della malattia, abbiamo recentemente scoperto che l&#39;esercizio fisico è più potente metodo fisiologico per indurre autofagia in un breve periodo di tempo 7, 8, 9. Rispetto fame, autofagia è effettivamente indotta da tapis roulant in esecuzione veloce come 30 min. Pertanto, l&#39;esercizio fisico è un approccio fisiologico comodo e potente per studiare il meccanismo di autofagia nel mediare benefici per la salute e la prevenzione delle malattie. Ci sono diversi marcatori proteici per la rilevazione di attività autofagia, tra cui LC3 e P62. LC3 (associata ai microtubuli proteina 1A / 1B-luce cHain 3) è una proteina citoplasmatica (LC3-formo) che viene coniugato con PE (fosfatidiletanolamina) su induzione autofagia. LC3 PE-lipidated (modulo LC3-II) viene reclutato su membrane autophagosomal e può essere utilizzato per visualizzare autophagosomes quando etichettati con GFP. La sua traslocazione dal citosol puntata strutture di autophagosomes al microscopio è un&#39;indicazione di induzione autofagia. p62 è un recettore di carico per supporti autofagia (come proteine ​​ubiquitinazione), ed è incorporato autophagosomes pure. Poiché la proteina è degradata in lisosomi insieme autophagosomes, i suoi livelli possono essere usati per misurare il flusso autofagia. Qui mostriamo come utilizzare questi marcatori di quantificare l&#39;autofagia in diversi tessuti di topo indotte da esercizio aerobico, compreso l&#39;esercizio forzato (tapis roulant) e esercizio volontario (in esecuzione ruote). Le stesse procedure possono essere applicate anche alla misurazione in vivo dell&#39;autofagia dopo il trattamento di altri induttori.

<table><tbody><tr><td>Treadmill</td><td>Columbus Instruments</td><td>150-RM Exer 3/6</td><td></td></tr><tr><td>Mouse running wheel</td><td>Super Pet</td><td>100079365</td><td>diameter 11.4 cm</td></tr><tr><td>Odometer</td><td>Bell</td><td>DASHBOARD 100</td><td></td></tr><tr><td>Syringe pump</td><td>KD Scientific</td><td>KDS100</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon</td><td>Model: inverted microscope ECLIPSE</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM 1850UV</td><td></td></tr><tr><td>Homogenizer</td><td>IKA</td><td>003737001 / Model: T10 Basic S1</td><td></td></tr><tr><td>Chloroquine</td><td>CAYMAN CHEMICAL COMPANY</td><td>14194</td><td></td></tr><tr><td>Parafolmaldehye</td><td>SIGMA-ALDRICH</td><td>P6148</td><td>Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer.</td></tr><tr><td>Mounting media</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>p62 antibody</td><td>BD Biosciences</td><td>610833</td><td></td></tr><tr><td>LC3 antibody</td><td>Novus Biologicals</td><td>NB100-2220</td><td></td></tr><tr><td>2x Laemmli Sample Buffer</td><td>Bio-Rad Laboratories</td><td>161-0737</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td></td></tr></tbody></table>

activating autophagy by aerobic exercise in mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Questo protocollo descrive due diversi metodi per indurre l&#39;autofagia nei tessuti di topo di esercizio aerobico: un totale di 90 minuti di esercizio fisico forzato su un tapis roulant a più corsie preceduto da due giorni di acclimatazione; o due settimane di esercizio volontario su una ruota in esecuzione usato dai topi alloggiati individualmente. In ogni protocollo di esercizio, siamo in grado di misurare il flusso autofagia da microscopia a fluorescenza e l&#39;analisi Western Blo...

Watch this Scientific Journal Video about Activating Autophagy by Aerobic Exercise in Mice at JoVE.com

Activating Autophagy by Aerobic Exercise in Mice

attivazione autofagia è utile nella prevenzione di varie malattie. Uno degli approcci fisiologiche per indurre autofagia in vivo è l&#39;esercizio fisico. Qui mostriamo come attivare l&#39;autofagia da esercizi aerobici e misurare i livelli di autofagia nei topi.

Attivazione di autofagia per esercizio aerobico nei topi

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

activating-autophagy-by-aerobic-exercise-in-mice

Research

JoVE Journal

Biology

12.2K Views.  Northwestern University.  attivazione autofagia è utile nella prevenzione di varie malattie. Uno degli approcci fisiologiche per indurre autofagia in vivo è l'esercizio fisico. Qui mostriamo come attivare l'autofagia da esercizi aerobici e misurare i livelli di autofagia nei topi. 

Video: Activating Autophagy by Aerobic Exercise in Mice

Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in na&#239;ve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.

A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters.

Valutazione della murini esercizio di resistenza senza l&#39;uso di una griglia Shock: Un&#39;alternativa alla Esercizio forzato

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most ...

Ricerca

Comportamento

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

Lipophagy is a selective form of autophagy that involves the degradation of lipid droplets. Dysfunctions in this process are associated with cancer development. However, the precise mechanisms are not yet fully understood. This protocol describes quantitative imaging approaches to better understand the interplay between autophagy, lipid metabolism, and cancer progression.

Esplorare la regolazione del catabolismo delle goccioline lipidiche attraverso la lipofagia

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This ...

Ricerca sul cancro

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro e In Vivo rilevamento di Mitophagy in cellule umane, C. Eleganse topi

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Medicina

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

Valutare il flusso autofagico misurando LC3, p62 e LAMP1 Co-localizzazione usando la citometria del flusso di imaging multispettrale

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Biologia

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Studio delle interazioni proteina-proteina nella ricerca di autofagia

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

In Situ Immunofluorescenti colorazione di autofagia nelle cellule staminali muscolari

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Il dosaggio di sequestro di lattato deidrogenasi — Un metodo semplice e affidabile per determinare l'attività autofagica sequestro Bulk in cellule di mammifero

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Flux-Based Assay for the Identification of Autophagy Modulators for Osteoarthritis

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify regulators of autophagy could be used for the identification of novel therapeutics. This article describes a cell-based imaging screening workflow developed to monitor autophagic flux using LC3 as a reporter of autophagic flux (mCherry-EGFP-LC3B) in human chondrocytes. Data acquisition is performed using an automated High Content Imaging Screening System microscope. An algorithm-based automated image analysis protocol was developed and validated to identify molecules activating autophagic flux. Critical steps, explanatory notes, and improvements over current autophagy monitoring protocols are reported. Physiologically relevant phenotypic screening approaches to target hallmarks of aging can facilitate more effective drug discovery strategies for age-related musculoskeletal diseases.

<meta charset="utf8"></head><body>Saggio basato sul flusso per l'identificazione di modulatori dell'autofagia per l'osteoartrite

This paper details monitoring autophagy flux to identify new molecules by cell-based imaging screening.

Saggio basato sul flusso per l'identificazione di modulatori dell'autofagia per l'osteoartrite

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Un modello di ruota da corsa semplice ed economico per l'allenamento di resistenza progressivo nei topi

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

Attivazione di autofagia per esercizio aerobico nei topi

Riepilogo

Esplora Altri Video

Valutazione della murini esercizio di resistenza senza l'uso di una griglia Shock: Un'alternativa alla Esercizio forzato

Esplorare la regolazione del catabolismo delle goccioline lipidiche attraverso la lipofagia

In Vitro e In Vivo rilevamento di Mitophagy in cellule umane, C. Eleganse topi

Valutare il flusso autofagico misurando LC3, p62 e LAMP1 Co-localizzazione usando la citometria del flusso di imaging multispettrale

Studio delle interazioni proteina-proteina nella ricerca di autofagia

In Situ Immunofluorescenti colorazione di autofagia nelle cellule staminali muscolari

Il dosaggio di sequestro di lattato deidrogenasi — Un metodo semplice e affidabile per determinare l'attività autofagica sequestro Bulk in cellule di mammifero

Saggio basato sul flusso per l'identificazione di modulatori dell'autofagia per l'osteoartrite

Saggio basato sul flusso per l'identificazione di modulatori dell'autofagia per l'osteoartrite

Un modello di ruota da corsa semplice ed economico per l'allenamento di resistenza progressivo nei topi