This work was supported in part by NIH grants (1R01CA137494, R01CA132115, R01CA086072 to R.G.P.), the Kimmel Cancer Center NIH Cancer Center Core grant P30CA056036 (R.G.P.), a grant from the Breast Cancer Research Foundation, generous grants from the Dr. Ralph and Marian C. Falk Medical Research Trust (R.G.P.) and a grant from the Pennsylvania Department of Health (R.G.P.). In part this work was supported by an American Italian Cancer Foundation postdoctoral fellowship (G.D.) and Bioimaging Shared Resource of the Sidney Kimmel Cancer Center (NCI 5 P30 CA-56036).The Department specifically disclaims responsibility for an analysis, interpretations or conclusions. There are no conflicts of interest associated with this manuscript.

1. elettroporazione di fibroblasti con entrambi vettori di espressione EYFP-Parkin e pDsRed2-Mito <ol><li> Far crescere le cellule di fibroblasti embrionali di topo immortalato su 10 centimetri piatto di tessuto-cultura con DMEM (modificata mezzo di Eagle di Dulbecco) medio supplementato con 10% di siero fetale bovino, 2 mmol / l L-glutammina, 100 U / ml di penicillina, e 100 mg / ml streptomicina in atmosfera umidificata contenente il 5% di CO 2 a 37 ° C. <ol><li> Alla confluenza cella 80%, scartare il mezzo DMEM completo per aspirazione sterile e aggiungere 10 ml di 1x sterile tampone fosfato (PBS) (80 g di NaCl, 2,0 g di KCl, 14,4 g di Na 2 HPO 4, 2,4 g di KH 2 PO 4 e 1 L distillata H 2 O, PH: 7.4). </li><li> Eliminare il PBS mediante aspirazione sterile e aggiungere 1 ml di tripsina-EDTA 0,25%. Incubare la piastra a 37 ° C finché le cellule vengono staccate (2 - 3 min). Aggiungere 4 ml di terreno DMEM completo, sospendere nuovamente le cellule ed estrarre 10 _6; l di sospensione cellulare per il conteggio emocitometro. NOTA: Il emocitometro è progettato in modo che il numero di cellule in un set di 16 quadrati angolo è equivalente al numero di cellule x 10 4 / ml. </li></ol></li><li> Seed 1 x 10 6 cellule su 10 cm piatti di coltura di tessuti 24 ore prima del processo di elettroporazione. </li><li> Eliminare il terreno completo DMEM per aspirazione sterile e aggiungere 10 ml di PBS sterile. Eliminare il PBS mediante aspirazione sterile e aggiungere 1 ml di tripsina-EDTA 0,25%. Incubare la piastra a 37 ° C finché le cellule vengono staccate (2 - 3 min). Aggiungere 4 ml di terreno DMEM completo e risospendere le cellule in una provetta da 15 ml. </li><li> Spin le celle in basso a 250 xg per 5 minuti usando un-centrifuga refrigerata (4 ° C). Eliminare il supernatante mediante aspirazione sterile e risospendere il pellet in 1 ml di PBS sterile. Spin le cellule giù a 250 xg per 5 minuti a 4 ° C. </li><li> Eliminare il surnatante tramite aspirazione sterili, e aggiungere 100 ml of soluzione mix per l&#39;elettroporazione (82 ml di soluzione elettroporazione V + 18 ml di soluzione supplementare 1) al pellet e risospendere delicatamente il pellet pipettando (Per maggiori dettagli fare riferimento alla guida per l&#39;utente). Aggiungere 2 mg di EYFP-Parkin (eccitazione / emissione 514/527) e 1 mg pDsRed2-Mito (eccitazione / emissione 565/620) alla sospensione cellulare. </li><li> Trasferire la soluzione nella cuvetta sterile utilizzando una Pasteur monouso (entrambi gli strumenti sono forniti con il kit) e l&#39;elettroporazione utilizzando il programma di pre-set NIH / 3T3 U-030 (impulso singolo, tensione a 200 V, capacità 960 uF, tempo di impulso 20 millisecondi , numero di impulsi: 1). </li><li> Subito dopo l&#39;elettroporazione, aggiungere 500 ml di mezzo fresco pre-riscaldato DMEM completo e inseminare la cella in 6 cm Piatto tessuto-coltura live-imaging grado e incubate in atmosfera umidificata contenente il 5% di CO 2 a 37 ° C per 24 ore. </li></ol> 2. Time-Lapse Video Microscopia <ol><li&gt; impostare la temperatura della camera del microscopio a 37 ° C prima dell&#39;uso. </li><li> A questo punto, preparare un supporto specifico imaging dal vivo di procedere con il protocollo sperimentale. Preparare il supporto di immagini dal vivo come segue; miscela DMEM privo di fenolo supplementato con 10% di siero fetale bovino, 2 mmol / l L-glutammina, 100 U / ml di penicillina, e 100 mg / ml di streptomicina. Pre-riscaldare il mezzo di imaging in tempo reale a 37 ° C in un bagno d&#39;acqua. </li><li> Eliminare il medium delle cellule elettroporate di aspirazione sterile e aggiungere 1 ml di terreno di immagini dal vivo pre-riscaldato, con una pipetta P1000 e incubare la piastra per 30 minuti a 37 ° C. </li><li> Durante il periodo di incubazione piatto, afflusso 5% di CO 2 nella camera già del microscopio ad una temperatura stabile di 37 ° C. </li><li> Posizionare la piastra con le cellule elettroporate nella camera del microscopio evitando i principali movimenti o oscillazioni. </li><li> Utilizzando l&#39;interfaccia software, impostare il microscopio per rilevare sia fluoresegnali scence dalle proteine ​​di fusione codificate dai vettori co-trasfettate, EYFP-Parkin (range eccitazione 495-510 nm e la gamma di emissione 520-550 nm, verde) e il pDsRed2-Mito (eccitazione massima 558 nm ed emissione massima 583 nm, rosso). <ol><li> Aprire il software di video microscopia time-lapse. Nel menu superiore selezionare il FITC per EYFP-Parkin e rodamina per pDsRed2-Mito. Nel menu superiore selezionare l&#39;ingrandimento (20X). </li></ol></li><li> Utilizzando l&#39;interfaccia del software, cercare la singola cellula che esprime entrambi i vettori co-trasfettate e registrare la posizione. Ripetere questa operazione fino a quando viene raccolto un minimo di 10 cellule per ogni condizione sperimentale (gruppo sperimentale). <ol><li> Selezionare il menu &quot;Applicazioni&quot; e cliccare su Multi Dimensional acquisizione. Nelle finestre Multi Dimensional Acquisizione selezionare i parametri necessari, quali il numero di acquisizioni, l&#39;intervallo di tempo tra ogni acquisizione e la posizione della cella registrata. Clicca su &quot;Acquisisci&quot;Per avviare il processo di acquisizione. </li></ol></li><li> Avviare l&#39;acquisizione basale sia EYFP-Parkin e segnale fluorescente DsRed2-Mito raccolta immagini ogni 5 min per un intervallo totale di 15 min. </li><li> Preparare pre-riscaldato mezzo di imaging in tempo reale con una concentrazione di FCCP due volte superiore alla concentrazione finale di lavoro (0,1 - 10 pM, dipendente dal tipo cellulare). </li><li> Interrompere il processo di acquisizione e aggiungere delicatamente 1 ml di pre-riscaldato medie immagini dal vivo con FCCP nella piastra all&#39;interno della camera del microscopio con una pipetta P1000. </li><li> Riavviare il processo di acquisizione come precedentemente descritto, per un periodo totale di 3 ore, utilizzando la posizione registrata. Salvare tutte le immagini acquisite per analizzare e creare un video del processo mitophagic quando l&#39;acquisizione è finita. </li></ol> 3. Analisi <ol><li> Raccogliere tutte le immagini acquisite in formato &quot;.tiff&quot; su un personal computer </li><li> Aprire le immagini con una grafica morbidaware e definire l&#39;intervallo temporale si è verificato dal mitocondriale indotta depolarizzazione con FCCP alla prima immagine che mostra il reclutamento di Parkin nella membrana mitocondriale (Le immagini sono acquisite ogni 5 minuti). Ripetere questa analisi per ogni cella nel gruppo sperimentale. </li><li> Etichettare la prima cella della colonna su un foglio con il nome sperimentale gruppo. Per ogni gruppo sperimentale, misurare gli intervalli temporali per Parkin assunzione di un minimo di 10 cellule. Fare una media degli intervalli temporali calcolati per Parkin reclutamento e definire la deviazione standard per ciascun gruppo sperimentale (Media ± SD, N = 10). <ol><li> Organizzare in colonne singoli intervalli temporali calcolati. Ogni colonna contiene il n = 10 misurazioni del gruppo sperimentale. </li><li> Selezionare la &quot;media&quot; funzione dal menu Formula Builder. Selezionare i dati nella colonna. Selezionare la funzione &quot;deviazione standard&quot; dal menu Formula Builder. Select i dati nella colonna. </li></ol></li><li> Usando un approccio statistico, confrontare i gruppi sperimentali che applicano la statistica appropriata per definire una differenza significativa nella dinamica mitophagy tra i gruppi sperimentali. (Per esempio, due gruppi = di Student t-test, tre o più gruppi = ANOVA più un test post-hoc) </li></ol>

<ol>
	<li>Youle, R. J., Narendra, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mitophagy.">Mechanisms of mitophagy.</a> Nat Rev Mol Cell Biol. 12, 9-14 (2011).</li><li>Zhi, X., Zhong, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+cancer.">Autophagy in cancer.</a> F1000Prime Rep. 7, 18 (2015).</li><li>Ding, W. X., Yin, X. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy:+mechanisms,+pathophysiological+roles,+and+analysis.">Mitophagy: mechanisms, pathophysiological roles, and analysis.</a> Biol Chem. 393, 547-564 (2012).</li><li>Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+mitochondrial+autophagy,+or+mitophagy,+as+a+targeted+defense+against+oxidative+stress,+mitochondrial+dysfunction,+and+aging.">Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging.</a> Rejuvenation Res. 8, 3-5 (2005).</li><li>Kissova, I., Deffieu, M., Manon, S., Camougrand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uth1p+is+involved+in+the+autophagic+degradation+of+mitochondria.">Uth1p is involved in the autophagic degradation of mitochondria.</a> J Biol Chem. 279, 39068-39074 (2004).</li><li>Kubli, D. A., Gustafsson, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+mitophagy:+the+yin+and+yang+of+cell+death+control.">Mitochondria and mitophagy: the yin and yang of cell death control.</a> Circ Res. 111, 1208-1221 (2012).</li><li>Redmann, M., Dodson, M., Boyer-Guittaut, M., Darley-Usmar, V., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+mechanisms+and+role+in+human+diseases.">Mitophagy mechanisms and role in human diseases.</a> Int J Biochem Cell Biol. 53, 127-133 (2014).</li><li>Di Sante, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+sirt1+promotes+prostatic+intraepithelial+neoplasia,+reduces+mitophagy,+and+delays+park2+translocation+to+mitochondria.">Loss of sirt1 promotes prostatic intraepithelial neoplasia, reduces mitophagy, and delays park2 translocation to mitochondria.</a> Am J Pathol. 185, 266-279 (2015).</li><li>Tanaka, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin-mediated+selective+mitochondrial+autophagy,+mitophagy:+Parkin+purges+damaged+organelles+from+the+vital+mitochondrial+network.">Parkin-mediated selective mitochondrial autophagy, mitophagy: Parkin purges damaged organelles from the vital mitochondrial network.</a> FEBS Lett. 584, 1386-1392 (2010).</li><li>Shiba-Fukushima, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1-mediated+phosphorylation+of+the+Parkin+ubiquitin-like+domain+primes+mitochondrial+translocation+of+Parkin+and+regulates+mitophagy.">PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy.</a> Sci Rep. 2, 1002 (2012).</li><li>Trinh, J., Farrer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+genetics+of+Parkinson+disease.">Advances in the genetics of Parkinson disease.</a> Nat Rev Neurol. 9, 445-454 (2013).</li><li>Murphy, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+mitochondria+produce+reactive+oxygen+species.">How mitochondria produce reactive oxygen species.</a> Biochem J. 417, 1-13 (2009).</li><li>Heytler, P. G., Prichard, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+class+of+uncoupling+agents--carbonyl+cyanide+phenylhydrazones.">A new class of uncoupling agents--carbonyl cyanide phenylhydrazones.</a> Biochem Biophys Res Commun. 7, 272-275 (1962).</li><li>Karan, G., Yang, Z., Zhang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+wild+type+and+mutant+ELOVL4+in+cell+culture:+subcellular+localization+and+cell+viability.">Expression of wild type and mutant ELOVL4 in cell culture: subcellular localization and cell viability.</a> Mol Vis. 10, 248-253 (2004).</li><li>Jin, S. M., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1-+and+Parkin-mediated+mitophagy+at+a+glance.">PINK1- and Parkin-mediated mitophagy at a glance.</a> J Cell Sci. 125, 795-799 (2012).</li><li>Kerr, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualisation+of+macropinosome+maturation+by+the+recruitment+of+sorting+nexins.">Visualisation of macropinosome maturation by the recruitment of sorting nexins.</a> J Cell Sci. 119, 3967-3980 (2006).</li><li>Edin, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-D+gel+culture+and+time-lapse+video+microscopy+of+the+human+vestibular+nerve.">3-D gel culture and time-lapse video microscopy of the human vestibular nerve.</a> Acta Otolaryngol. 134, 1211-1218 (2014).</li><li>Ramsden, A. E., Mota, L. J., Munter, S., Shorte, S. L., Holden, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SPI-2+type+III+secretion+system+restricts+motility+of+Salmonella-containing+vacuoles.">The SPI-2 type III secretion system restricts motility of Salmonella-containing vacuoles.</a> Cell Microbiol. 9, 2517-2529 (2007).</li><li>Meseguer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+incubation+and+selection+in+a+time-lapse+monitoring+system+improves+pregnancy+outcome+compared+with+a+standard+incubator:+a+retrospective+cohort+study.">Embryo incubation and selection in a time-lapse monitoring system improves pregnancy outcome compared with a standard incubator: a retrospective cohort study.</a> Fertil Steril. 98, 1481-1489 (2012).</li><li>Campbell, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+a+risk+classification+of+aneuploidy+in+human+embryos+using+non-invasive+morphokinetics.">Modelling a risk classification of aneuploidy in human embryos using non-invasive morphokinetics.</a> Reprod Biomed Online. 26, 477-485 (2013).</li><li>Bingol, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+deubiquitinase+USP30+opposes+parkin-mediated+mitophagy.">The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy.</a> Nature. 510, 370-375 (2014).</li><li>Carroll, R. G., Hollville, E., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin+sensitizes+toward+apoptosis+induced+by+mitochondrial+depolarization+through+promoting+degradation+of+Mcl-1.">Parkin sensitizes toward apoptosis induced by mitochondrial depolarization through promoting degradation of Mcl-1.</a> Cell Rep. 9, 1538-1553 (2014).</li><li>Batlevi, Y., La Spada, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+autophagy+in+neural+function,+neurodegenerative+disease,+neuron+cell+death,+and+aging.">Mitochondrial autophagy in neural function, neurodegenerative disease, neuron cell death, and aging.</a> Neurobiol Dis. 43, 46-51 (2011).</li><li>Frank, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+is+triggered+by+mild+oxidative+stress+in+a+mitochondrial+fission+dependent+manner.">Mitophagy is triggered by mild oxidative stress in a mitochondrial fission dependent manner.</a> Biochim Biophys Acta. 1823, 2297-2310 (2012).</li><li>Neumann, E., Schaefer-Ridder, M., Wang, Y., Hofschneider, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields.">Gene transfer into mouse lyoma cells by electroporation in high electric fields.</a> EMBO J. 1, 841-845 (1982).</li><li>Sugar, I. P., Neumann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+model+for+electric+field-induced+membrane+pores.+Electroporation.">Stochastic model for electric field-induced membrane pores. Electroporation.</a> Biophys Chem. 19, 211-225 (1984).</li><li>Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variants+of+mitochondrial+autophagy:+Types+1+and+2+mitophagy+and+micromitophagy+(Type+3).">Variants of mitochondrial autophagy: Types 1 and 2 mitophagy and micromitophagy (Type 3).</a> Redox Biol. 2, 749-754 (2014).</li><li>Aggarwal, B. B., Quintanilha, A. T., Cammack, R., Packer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage+to+mitochondrial+electron+transport+and+energy+coupling+by+visible+light.">Damage to mitochondrial electron transport and energy coupling by visible light.</a> Biochim Biophys Acta. 502, 367-382 (1978).</li><li>Alexandratou, E., Yova, D., Handris, P., Kletsas, D., Loukas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fibroblast+alterations+induced+by+low+power+laser+irradiation+at+the+single+cell+level+using+confocal+microscopy.">Human fibroblast alterations induced by low power laser irradiation at the single cell level using confocal microscopy.</a> Photochem Photobiol Sci. 1, 547-552 (2002).</li><li>Kim, I., Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+selectively+degrades+individual+damaged+mitochondria+after+photoirradiation.">Mitophagy selectively degrades individual damaged mitochondria after photoirradiation.</a> Antioxid Redox Signal. 14, 1919-1928 (2011).</li><li>Coutu, D. L., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+cellular+processes+by+long-term+live+imaging--historic+problems+and+current+solutions.">Probing cellular processes by long-term live imaging--historic problems and current solutions.</a> J Cell Sci. 126, 3805-3815 (2013).</li></ol>

The authors have nothing to disclose.

microscopia time-lapse può essere definita come la tecnica che si estende cellule vive da una singola osservazione in tempo per l&#39;osservazione delle dinamiche cellulari per lunghi periodi di tempo. Questa metodologia è distinto da un semplice confocale microscopia cellule in vivo perché permette all&#39;osservatore di identificare in tempo reale una singola proteina fluorescente-tag e seguire la sua dinamica all&#39;interno di una singola cella vivo. Infatti, la microscopia confocale può facilmente identifica proteine ​​immuno-marcato utilizzando anticorpi fluorescenti, ma non consente osservando la cella e l&#39;evento molecolare nel suo ambiente vivo. Il microscopio video time-lapse è utilizzato principalmente nella ricerca monitoraggio dei processi molecolari in diversi tipi di cellule, come HEK293 16, neuroni primari 17 e 18 cellule HeLa, ma ha anche l&#39;applicazione clinica come la gravidanza e la previsione aneuploidie 19,20. Time-lapse microscopia fornisce embryologists con ulteriori informazioni sullo sviluppo embrionale per una gravidanza clinica, ed è possibile che queste informazioni possono essere utilizzate per migliorare la nostra capacità di selezionare gli embrioni vitali per il trasferimento. Ulteriori vantaggi dell&#39;uso di microscopia time-lapse includono meno la manipolazione degli embrioni e dei loro piatti per osservazioni intermedie, che ridurrebbe il rischio di perdita o contaminazione all&#39;interno del laboratorio 19. Le cellule sono in grado di mantenere un sano omeostasi mitocondriale degradando mitocondri danneggiati e disfunzionali attraverso mitophagy. Mitophagy difettoso è legata alla malattia di Parkinson 21, l&#39;apoptosi 22, invecchiamento e cancro 23 24. Mitophagy è guidato dalla ubiquitina ligasi Parkin e PINK1 21. Data l&#39;importanza di questo processo per mantenere uno status mitocondriale sano, abbiamo trovato importante per studiare la dinamica di questo evento con un video microscopia time-lapse. Il pro propostoProtocollo fornirà la prova di come i mitocondri omeostasi è influenzato in diverse condizioni di cellulari, compresi i cambiamenti nelle funzioni di espressione genica e di proteine. In questo studio, abbiamo trasfettate fibroblasti mediante elettroporazione. Il metodo elettroporazione applica un impulso elettrico ad una tensione ottimale della durata di alcuni microsecondi alla sospensione cellulare per aumentare la permeabilità della membrana cellulare disturbando il doppio strato fosfolipidico della membrana. Questa azione provoca la formazione di pori temporanei. La permeabilizzazione della membrana consente il DNA da introdurre nella cella 25. Abbiamo scelto questo processo trasfezione transitoriamente fibroblasti in modo da avere una maggiore efficienza trasfezione di entrambi i plasmidi EYFP-Parkin e pDsRed2-Mito. Infatti, il processo di elettroporazione è circa dieci volte più efficace di trasfezione chimica 26. Il processo di acquisizione immagini possono essere compromesso da entrambi exteFattori rnal; sbalzi di temperatura, vibrazioni, umidità e fattori interni; pH, la motilità cellulare. L&#39;elettroporazione e il monitoraggio fluorescenti sono due passaggi considerato il più critico per il successo di questo protocollo sperimentale. Anche se l&#39;efficienza di trasfezione è superiore ad altri metodi, l&#39;elettroporazione uccide ~ 20% delle cellule elettroporate. Un&#39;altra questione importante è la qualità di acquisizione delle immagini durante la fase di monitoraggio. Poiché l&#39;ambiente cellulare è dinamico, il piano di messa a fuoco deve continui aggiustamenti per mantenere l&#39;attenzione durante il processo di acquisizione. danno mitocondriale può essere causato da membrane depolarizzazione mitocondriale e lo stress ossidativo. Questi fattori inducono una risposta mitophagic. La prova che i mitocondri depolarizzazione può indurre mitophagy selettivo viene da esperimenti fotodanneggiamento che portano alla produzione di ROS e lesioni mitocondriale 27-30. Al fine di indurre mem mitocondrialedepolarizzazione brane e la risposta mitophagic, FCCP è stato utilizzato come protonophore 10. FCCP è uno ionoforo che permette ai protoni di attraversare doppi strati lipidici che inducono depolarizzazione della membrana. Usando questo approccio abbiamo indotto mitophagy e seguito la dinamica del marcatore molecolare. La concentrazione di FCCP è fondamentale, in quanto può essere citotossici e promuovere la morte cellulare. Poiché questo approccio si basa sull&#39;osservazione delle cellule viventi, la concentrazione finale di FCCP deve essere regolata in modo dipendente tipo cellulare per indurre mitophagy senza innescare i meccanismi che portano alla morte cellulare. Nel complesso, riteniamo che a seguito della dinamica di mitophagy, utilizzando EYFP-Parkin come un marcatore fluorescente, fornirà nuovi elementi in funzione mitocondriale. La rimozione selettiva dei mitocondri tramite mitophagy è un processo cruciale nel mantenimento della qualità mitocondriale, la vitalità cellulare e l&#39;omeostasi. Questo approccio immagini dal vivo sarà luilp per comprendere il processo di mitophagic in condizioni sane e stressate. approcci moderni stanno estendendo ulteriormente time-lapse osservazioni al microscopio di là di fare film di dinamiche cellulari. Sempre questo confine è sfocata, come tecniche di citometria vengono integrati con tecniche di imaging per il monitoraggio e la misurazione delle attività dinamiche di cellule e strutture subcellulari 31.

Macro autophagy is an intracellular process that involves the catabolic degradation of both damaged and dysfunctional cellular components, such as organelles and proteins for the purpose of either recycling or energy production. To initiate this metabolic process, the cell engulfs the damaged cellular components into a double-membrane structure, known as an autophagosome, which fuses with a lysosome and its content is degraded and recycled 1,2. There are two major types of autophagy, the non-selective and selective. The non-selective autophagy process occurs when the cell is under nutrient deprivation conditions and needs to scavenge for both essential nutrients and energy. However, selective autophagy occurs to mediate the removal of both dysfunctional/damaged organelles and proteins that otherwise could be toxic. One of the most studied selective autophagy process is the removal of mitochondria, termed mitophagy 1,3-5.
Mitochondria are the central organelles for cell metabolism and the primary source of adenosine triphosphate (ATP) via oxidative phosphorylation through the electron transport chain, fatty acid oxidation, and tricarboxylic acid (TCA) cycle. Moreover, mitochondria regulate reactive oxygen species (ROS) production and release proteins that participate in cell death pathways 6-8.
PTEN-induced putative kinase 1 (PINK1) and Parkin RBR E3 ubiquitin ligase (Parkin) are the key proteins implicated in the mitophagy process. Parkin can protect against cell death by keeping the cell healthy through mitochondrial quality control9. Upon the loss of mitochondrial membrane potential, cytosolic Parkin is recruited to the mitochondria by PINK1. This recruitment triggers the sequential events of mitophagy 10. There is a broad range of evidence that mitophagy is a fundamental mitochondria quality control process and abnormalities in this process drive disease 7. For instance, autosomal recessive Parkinson's disease has been associated with mutations in the genes that encode for Parkin and PINK1 (PARK2 and PINK1, respectively) 11. The quality control of mitochondrial health is essential for the removal of mitochondria that contribute to the accumulation of ROS12. Excessive presence of intracellular ROS can lead to damage of both nuclear and mitochondrial DNA (DNA and mt DNA, respectively).
Herein, we show a time-lapse video microscopy approach to follow the aggregation of Parkin after the induction of Parkin-mediated mitophagy in immortalized mouse embryonic fibroblasts via in vitro administration of carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP), an uncoupling agent. FCCP disrupts ATP synthesis by short circuiting protons across the outer mitochondria membrane and hence uncoupling oxidative phosphorylation from the electron transport chain 13. Triggering the depolarization of the mitochondrial membrane leads to the disruption of mitochondria and selective Parkin-dependent removal. Therefore, transfecting the cells of interest with an expression vector encoding Parkin fused with a fluorescent marker (enhanced yellow fluorescent protein, EYFP) can be used as a fluorescent tag to follow the recruitment of Parkin during the mitophagic process. In order to visualize the mitochondria, we co-transfected pDsRed2-Mito, which encodes red fluorescent protein (DsRed2) that contains a mitochondrial targeting sequence of cytochrome c oxidase subunit VIII (Mito). pDsRed2-Mito is designed for fluorescent labeling of mitochondria14. The time required for Parkin translocation into the mitochondrial membrane can be measured and gives an indirect measure of cellular health. For example, we can say that if a cell line knocked-out for a particular gene of interest shows either a faster or slower recruitment of Parkin after the induction of mitophagy by FCCP, that gene product would be a key player in order to keep the metabolic rates of the cell at the physiological status and prevent the development of diseases. Therefore, the time-lapse video microscopy provides a very powerful tool for both basic and clinical research applications in following the dynamic of labeled proteins during their molecular processes and understanding how these processes are affected during a pathological condition.

<table border="0" cellpadding="0" cellspacing="0" width="1736">
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">DMEM</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">10-013-CV</td>
			<td style="width:369px;">Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">PHENOL-FREE DMEM</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">17-205-CV</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">FETAL BOVINE SERUM</td>
			<td style="width:357px;">Sigma-Aldrich</td>
			<td style="width:276px;">F2442</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">L-GLUTAMINE</td>
			<td style="width:357px;">Gibco</td>
			<td style="width:276px;">25030</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">PENICILLIN/ STREPTOMYCIN</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">30-002-CI</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">EYFP-PARKIN EXPRESSION VECTOR</td>
			<td style="width:357px;">Addgene</td>
			<td style="width:276px;">23955</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">pDsRed2-Mito EXPRESSION VECTOR</td>
			<td style="width:357px;">Clontech</td>
			<td style="width:276px;">632421</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">NUCLEOFECTOR 2B DEVICE</td>
			<td style="width:357px;">LONZA</td>
			<td style="width:276px;">AAD-1001S</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">NUCLEOFECTOR FOR KIT R NIH/3T3</td>
			<td style="width:357px;">LONZA</td>
			<td style="width:276px;">VCA-1001</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">ZEISS AXIOVERT 200M INVERTED MICROSCOPE</td>
			<td style="width:357px;">CARL ZEISS</td>
			<td style="width:276px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Carbonyl Cyanide 4-(trifluoromethoxy)-Phenylhydrazone (FCCP)</td>
			<td style="width:357px;">Sigma-Aldrich</td>
			<td style="width:276px;">C2920</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">MetaMorph</td>
			<td style="width:357px;">Molecular Devices</td>
			<td style="width:276px;">Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:735px;">ImageJ</td>
			<td style="width:357px;">National Institute of Health</td>
			<td style="width:276px;">Experimental Builder</td>
			<td></td>
		</tr>
	</tbody>
</table>

time-lapse video microscopy for assessment of eyfp-parkin aggregation as a marker for cellular mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Qui, si mostra come il video time-lapse microscopia è una tecnica potente che può essere utilizzato per seguire gli eventi molecolari di proteine ​​fluorescenti-taggato in una singola cella. I risultati rappresentativi mostrano anche come questa tecnica permette l&#39;acquisizione di immagini di alta qualità. Quando le immagini del processo molecolare, si ottengono, abbiamo la possibilità di analizzare in modi diversi. Qui, analizziamo l&#39;intervallo di tempo tra l&#39;inizio del processo mitophagy indotta, che è il processo molecolare cruciale che mantenere l&#39;omeostasi cellulare rimozione selettiva dei mitocondri danneggiati. Questa tecnica fonda una vasta gamma di applicazioni. Può essere utilizzato per monitorare macro processo cellulare come migrazione cellulare o eventi molecolari micro attraverso l&#39;etichettatura delle molecole chiave coinvolte nel processo che deve essere studiato. o: keep-together.within-page = &quot;1&quot;&gt; Parkin-mediata Mitophagy La rimozione selettiva Parkin-mediata del processo mitocondri danneggiati e disfunzionale è riassunta nella figura 1, dove una depolarizzazione della membrana indotta dall&#39;agente disaccoppiamento FCCP innesca selettivo mitophagy Parkin-mediata, reclutando PINK1 alla membrana mitocondriale esterna. In seguito, Parkin è reclutato per i mitocondri danneggiati e interagisce con PINK1. Questo complesso Parkin-PINK1 porta alla ubiquitinazione del organello danneggiato e la sua integrazione in un autofagosoma, che si fonde con un lisosoma. Questo processo noto come mitophagy è considerato un processo metabolico cruciale al fine di mantenere l&#39;ambiente cellulare sano e permettere la rigenerazione dei mitocondri funzionali 8,15. protocollo sperimentale nt &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; Figura 2 mostra una rappresentazione schematica del protocollo sperimentale e il microscopio set-up utilizzato per seguire il reclutamento di EYFP-Parkin prima e dopo il processo mitophagy FCCP-indotta. Parkin reclutamento di depolarizzato membrana mitocondriale Nella Figura 3A, mostriamo la distribuzione Parkin amministrazione prima e dopo l&#39;FCCP. Durante i punti di tempo basali (BT5, Bt10 e BT15 min) EYFP-Parkin (verde) è omogeneamente diffuso in tutto il cellulare e la rete di mitocondri (rosso) sembra essere ben interconnessa. A seguito di somministrazione FCCP (FT = 0 min) (Figura 3B), osserviamo mitocondri frazionamento (FT = 5 min). Tuttavia, EYFP-Parkin è ancora omogeneamente diffuso anche se fuori il citoplasma. Mitophagy è stimolato a amministrazione postale FCCP 55 min,EYFP-Parkin è reclutato a livello delle membrane mitocondriali danneggiate per innescare il processo mitophagy (FT = 55). <img alt="Figura 1" src="/files/ftp_upload/53657/53657fig1.jpg" /> Figura 1. Rappresentazione schematica di Parkin-mediata Mitophagy. Una diminuzione del potenziale di membrana (Ψm) si verifica nei mitocondri danneggiati o disfunzionali e può essere indotta dall&#39;agente disaccoppiamento FCCP. La perdita di potenziale di membrana innesca selettivo mitophagy Parkin-mediata, reclutando PINK1 (rosso) alla membrana mitocondriale esterna. Parkin (blu) viene reclutato per i mitocondri danneggiati interagendo direttamente con PINK1. La presenza di Parkin porta alla ubiquitinazione (giallo) del organello danneggiata e la sua integrazione in un autofagosoma, che si fonde con un lisosoma. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53657/53657fig1large.jpg" target="_blank">Fai clic qui per vedere una versione più grande di questa figura</a>. <img alt="figura 2" src="/files/ftp_upload/53657/53657fig2.jpg" /> Figura 2. Microscopio Set-up e Rappresentazione del protocollo sperimentale. (A) Panoramica degli eventi sperimentali descritte nella sezione del protocollo. Gli eventi sperimentali riportati consentono all&#39;operatore di transitoriamente co-trasfezione delle cellule con EYFP-Parkin e pDsRed2-Mito mediante elettroporazione, al fine di seguire il processo mitophagy FCCP indotta utilizzando un (B) di set-up per il time-lapse approccio di video microscopia. BT = punto di tempo basale; FT = FCCP punto di tempo. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53657/53657fig2large.jpg" target="_blank">Fate clic qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53657/53657fig3.jpg" /> Figura 3. fibroblasti cellulari che mostrano Parkin Recruitme<html> nt dopo membrana mitocondriale depolarizzazione indotta da EYFP-Parkin (verde) traslocazione nei mitocondri (rosso) FCCP. è stato monitorato da microscopio video time-lapse durante il punto di tempo basale (BT) e inviare amministrazione FCCP (FT). Da BT = 0, EYFP-Parkin e fluorescenza mitocondriale sono stati rilevati ogni 5 minuti. (A) EYFP-Parkin è omogeneamente diffuso in tutto il tutto il periodo basale e la rete mitocondriale è intatto (frecce bianche). (B) frazionamento mitocondriale a causa di depolarizzazione della membrana da FCCP è registrato a FT = 5 min (frecce bianche) e il reclutamento EYFP-Parkin alle membrane mitocondriali danneggiate iniziato a FT = 55 min (frecce bianche). Le caselle bianche rappresentano l&#39;area ingrandita dei pannelli (5X). Per maggiori dettagli riguardanti l&#39;applicazione di questo protocollo sperimentale riferimento a riferimento 8. (Bar Scala: 10 micron).&gt; Clicca qui per vedere una versione più grande di questa figura.

Watch this Scientific Journal Video about Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy at JoVE.com

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-Lapse Video Microscopia per la valutazione di EYFP-Parkin aggregazione come marcatore per cellulare Mitophagy

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

time-lapse-video-microscopy-for-assessment-eyfp-parkin-aggregation-as

Universidad San Sebastian

Research

JoVE Journal

Biology

7.1K Views.  Thomas Jefferson University, SKCC. Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Video: Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

Monitoraggio Actina Smontaggio con Time-lapse Microscopia

Ricerca

Biologia

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse imaging di mitosi dopo la trasfezione di siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Un saggio microscopia a fluorescenza per Mitophagy monitoraggio nel lievito Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Microscopia time-lapse di Embriogenesi All&#39;inizio del Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Analisi quantitativa della migrazione casuale di celle usando Time-lapse Microscopia Video

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

L&#39;uso della microscopia Time lapse di visualizzare anossia indotta Suspended Animation in<em&gt; C. elegans</em&gt; Embrioni

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualizzazione di sviluppo craniofacciale nei SOX10: Kaede transgenici Zebrafish line utilizzando Time-lapse Microscopia confocale

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

Metodi di studio variazioni inerenti l'aggregazione proteica con l'età in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Microscopia confocale rivela l'aggregazione di recettore di superficie delle cellule tramite spettroscopia di correlazione di immagine

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

Un metodo Versatile per il montaggio di Arabidopsis lascia per l'Imaging di time-lapse videomicroscopia

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...