This work was supported by a National Institutes of Health grant (GM77311) and a seed grant from The Ohio Cancer Research Associates (to H.A.F.). SM was partially supported by an Up on the Roof fellowship from the Human Cancer Genetics Program of The Ohio State University Comprehensive Cancer Center.

Cultura 1. Cella <ol><li> Utilizzare telomerasi umana trascrittasi inversa (hTERT) cellule epiteliali del pigmento retinico immortalati (hTERT-RPE1, di cui qui RPE1). NOTA: le cellule RPE1 sono quasi diploide, non trasformati cellule umane che sono comunemente usati per studiare l&#39;assemblaggio centriolo e ciliogenesis. Queste cellule seguono un normale ciclo di duplicazione centriolo coordinato con un ciclo cellulare regolamentato. </li><li> Passaggio un quasi confluenti 100 millimetri piatto di coltura cellulare delle cellule RPE1 a diluizione 1: 5 della coltura originale in un piatto dolce 100 mm avente 10 ml di DME / F-12 (1: 1) media supplementato con 10% siero fetale bovino (FBS), 100 U / ml di penicillina G e 100 ug / ml di streptomicina (terreno completo è fatto riferimento come DME / F-12 di media). Grow cellule a 37 ° C in presenza di 5% CO 2. <ol><li> Incubare 12 millimetri rotondi vetrini in 50 ml di HCl 1 N in un bicchiere di vetro coperto, O / N senza agitazione, a 60 ° C in un bagno d&#39;acqua o incubatore. </li><li> After scartando la soluzione di acido, lavare i coprioggetti tre volte in 100 ml di acqua distillata incubando per 15 min, con agitazione intermittente. Ripetere il lavaggio simile in 70% etanolo e il 95% di etanolo. </li><li> Asciugare i coprioggetti dai loro diffusione singolarmente su una carta assorbente da laboratorio in un armadio biosicurezza, seguita da sterilizzazione con radiazioni UV per 60 min. </li></ol></li><li> Mettere 2-4 coprioggetto secco in un piatto di coltura cellulare di 35 mm. Diluire la soluzione di fibronectina o fibronectina come polimero ingegnerizzato (concentrazione archivio di 1 mg / ml) ad una concentrazione di 25 ug / ml in PBS coltura cellulare. <ol><li> Mettere 80-100 ml di soluzione diluita su ogni coprioggetto e incubare per 60 min per rivestire il lato superiore del vetrino. Lavare i coprioggetti tre volte con PBS prima di aggiungere terreno completo. </li></ol></li></ol> 2. crescita delle cellule e cellule Trattare con Proteasome Inhibitors <ol><li> Passaggio 2 x 10 5 asincrono crescerecellule ing RPE1 in ogni piatto 35 millimetri contenente coprioggetto e far crescere le cellule in 2 ml terreno completo. Sostituire il terreno di coltura ogni 24 ore con terreno completo fresco pre-riscaldato. <ol><li> Per analizzare l&#39;effetto di inibizione del proteasoma a livello centrosomica della proteina di prova (qui VDAC3 o γ-tubulina), crescono le cellule in due 35 piatti mm per 44 ore. Sostituire il terreno di coltura con terreno completo e aggiungere sia MG115 o DMSO come solvente comando ad una concentrazione finale di 5 mM o 0,05% rispettivamente. Allo stesso tempo, aggiungere BrdU alle cellule ad una concentrazione finale di 40 mM e incubare cellule per 4 ore. </li><li> Trasferire ogni coprioggetto in un 24-pozzetti. Aggiungere metanolo 500 microlitri refrigerata a ciascun pozzetto e incubare la piastra a -20 ° C per 10 minuti per fissare le cellule sui vetrini. Lavare immediatamente i vetrini tre volte con 500 microlitri tampone di lavaggio (PBS 1x contenente 0,5 mM MgCl 2 e il 0,05% Triton-X 100). </li></ol></li><li> Raccogliere il residuocellule dal piatto da 35 mm e centrifugare le cellule a 1000 xg per 5 min. Utilizzare il pellet di cellule per analizzare le proteine ​​mediante western blotting (punto 6). </li></ol> 3. BrdU Pulse e Chase test per analizzare i livelli di proteine ​​nelle diverse fasi del ciclo cellulare <ol><li> Per analizzare la variazione del livello di una proteina (qui VDAC3, SAS6 o Cep135) a centrosomes nelle diverse fasi del ciclo cellulare, crescere le cellule in due piatti 35 mm contenenti coprioggetti per 44 hr. Sostituire il terreno di coltura con terreno completo contenente 40 mM BrdU e incubare le cellule per 4 ore nell&#39;incubatrice coltura cellulare. </li><li> Da un piatto, di trasferire i coprioggetti di un 24-pozzetti per fissare le cellule utilizzando metanolo freddo come descritto al punto 2.1.2. </li><li> Dopo l&#39;impulso di BrdU 4 ore, rimuovere il terreno-BrdU contenente, lavare le cellule una volta con PBS ed una volta con mezzo completo, aggiungere mezzo fresco, e poi far crescere le cellule in assenza di BrdU per vari tempi (tipicamente un&#39;altra 4 hrper le cellule RPE1) prima che fissa le cellule come descritto al punto 2.1.2. NOTA: Per le cellule RPE1, la maggior parte delle cellule BrdU-positive sono in ritardo S o G2-fase, dopo un inseguimento 4 ore. Questo deve essere determinata in modo indipendente per ciascun tipo di cellula. </li></ol> 4. Immunostaining <ol><li> Incubare le cellule fissate su vetrini in 200 microlitri di tampone bloccante (2% BSA e 0,1% TritonX-100 in PBS 1x) per 30 min. <ol><li> Incubare le cellule con una miscela di anticorpi primari (tipicamente un risalto in coniglio e un&#39;altra sollevato nel topo) diluito in tampone di bloccaggio O / N a 4 ° C in una camera umidificata. </li><li> Per fare una camera umidificata, mettere un tovagliolo di carta bagnata nella metà inferiore di un 1.000 ml box puntale vuota. Disporre una striscia di Parafilm sulla superficie cremagliera, e individuare una goccia (15-20 ml) della soluzione di anticorpi sulla Parafilm per ogni coprioggetto da incubate. Invertire un coprioggetto su una goccia di soluzione di anticorpi (in modo che le cellule sono immerse) e vicinoil coperchio della scatola punta. </li><li> Capovolgere nuovamente coprioggetto e tornare al piatto 24-bene. Lavare coprioggetto e poi incubare 150 miscela anticorpo secondario microlitri (qui green-colorante fluorescente coniugato anti-coniglio e colorante rosso fluorescente coniugato anti-topo diluito in tampone di bloccaggio) per 1 ora a temperatura ambiente. Lavare i coprioggetti tre volte. NOTA: Gli anticorpi secondari fluoroforo-coniugato sono fotosensibili. Proteggere i campioni dalla luce durante le fasi 4.1.3-5.1.2. </li></ol></li><li> Preparatevi per la colorazione anti-BrdU fissando cellule RPE1 colorate di nuovo con 500 microlitri metanolo raffreddato a -20 ° C per 10 minuti, come descritto al punto 2.1.2. Lavare i coprioggetti tre volte. NOTA: Questa fissazione assicura l&#39;etichettatura anticorpo primario e secondario della proteina (s) di interesse durante il processo di idrolisi acida nel passaggio seguente. <ol><li> Incubare le cellule in 200 ml di 2 N HCl per 30 minuti a RT. Neutralizzare con 300 ml di 1 M Tris-Cl, pH 8 und lavare le cellule tre volte con tampone di lavaggio. </li><li> Bloccare le cellule nuovamente utilizzando 200 microlitri di tampone bloccante per 30 minuti a RT. </li><li> Incubare le cellule con anticorpo anti-topo (BrdU diluito in tampone di bloccaggio) per 45 min a 37 ° C in una camera umidificata. Restituisce i coprioggetti torna al piatto 24 pozzetti, lavarli e poi incubare con blu-colorante fluorescente coniugato anti-rat anticorpo secondario (diluito in 150 ml di soluzione bloccaggio in piatto 24 pozzetti per 1 ora a temperatura ambiente. </li></ol></li><li> Lavare i coprioggetti tre volte. Spot una goccia (circa 3-6 ml) di una soluzione di montaggio contenente antifade reattivo su un vetrino da microscopio in vetro. Invertire un coprioggetto, con la cellula-lato rivolto verso il basso, sulla soluzione di montaggio. Pulire il liquido in eccesso premendo delicatamente il vetrino contro il vetrino utilizzando tessuti di pulizia molli. Applicare lo smalto trasparente lungo il bordo del vetrino per sigillarlo sul vetrino da microscopio. </li></ol> 5. immunofluorescenza Immagine Acquisition e analisi <ol><li> Utilizzare un obiettivo ad immersione 100X Plan Apo (con un&#39;apertura numerica 1.4) per acquisire le immagini di cellule RPE1 BrdU-positive a temperatura ambiente. <ol><li> Mettere un vetrino sul microscopio (vedi attrezzature) collegato con una macchina fotografica in grado di digital imaging. Per ciascun fluoroforo, determinare il tempo di esposizione appropriata (tipicamente tra 300-1,000 msec) esaminando manualmente tutti i campioni di un esperimento. Prima di acquisire immagini di una cella, determinare manualmente all&#39;inizio appropriato e piano focale inferiore lungo l&#39;asse Z (tipicamente 0,2 micron passo). Acquisire le immagini di diversi campioni che si trovano su vetrini separati con tempo di esposizione identico per i diversi fluorofori lungo l&#39;asse Z utilizzando un pacchetto software di imaging digitale di microscopia. </li></ol></li><li> Eseguire deconvoluzione (in questi casi utilizzando n algoritmo prossimo) di tutte le pile di immagine acquisiti lungo l&#39;asse Z. <ol><li> Ottenere la proiezione intensità totale di ogni pila di immagini insiemeZ. </li></ol></li><li> Nelle cellule dove centrosomi sono vicini (distanza tra due centrosomi è inferiore a 2 micron), disegnare una piccola piazza (tipicamente 20-30 pixel per lato) intorno ad entrambi i centrosomi e segnare l&#39;area selezionata. <ol><li> Disegnare un quadrato più grande (in genere 24-35 pixel per lato) che circondano la prima piazza e segnare l&#39;area selezionata del piazzale. </li><li> Ottenere la zona (A) e l&#39;intensità di fluorescenza totale (F) di ciascun fluoroforo in ciascuna casella (S denota piccola scatola e L indica grande scatola). </li><li> Analizzare il fondo corretta intensità di fluorescenza di ciascun fluoroforo utilizzando la formula descritta da Howell et al. 29: F = F S - [(F L - F S) x A S / (A L - A S)]. </li><li> Ottenere l&#39;intensità di fluorescenza normalizzata della proteina di interesse (qui VDAC3 o SAS6) calcolando il rapporto tra l&#39;intensità di fluorescenza di fondo corretto dei suoi fluorophore a quella del fluoroforo utilizzato per lo standard prescelto interno (qui γ-tubulina, SAS6 o Cep135). </li></ol></li><li> Nel caso di analisi cellule dove centrosomes sono ben separati (distanza tra due centrosomi è maggiore di 2 micron), analizzare ogni centrosome separatamente disegnando due gruppi separati di scatole. Disegnare una piccola piazza (tipicamente 15-25 pixel per lato) intorno ad ogni centrosomi e segnare l&#39;area selezionata. Disegnare un quadrato più grande (20-30 pixel per lato) che circondano la prima piazza e segnare l&#39;area selezionata. <ol><li> Ottenere la zona (A) e l&#39;intensità di fluorescenza totale (F) di ciascun fluoroforo in ogni casella, e calcolare correzione del fondo fluorescenza intensità di ciascun fluoroforo, separatamente per ciascuna centrosome, come descritto al punto 5.3.3. </li><li> Combinare correzione del fondo fluorescenza intensità di ciascuna proteina dai due centrosomi per ottenere il livello centrosomica totale di proteine ​​che in quella cella. </li><li> Ottenere ilintensità normalizzata per la proteina di interesse calcolando il rapporto tra l&#39;intensità totale centrosomica all&#39;intensità centrosomica totale dello standard interno. </li></ol></li><li> Analizzare almeno 15-25 cellule per ogni condizione sperimentale e tracciare il grafico in un foglio di calcolo. </li></ol> 6. Analizzando il Proteine ​​totali Utilizzando Western Blotting <ol><li> Risospendere le cellule in radioimmunoprecipitazione dosaggio (RIPA) tampone contenente 50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Nonidet P-40, 1% desossicolato di sodio, 0,1% sodio dodecil solfato (SDS) e incubare per 10 min in ghiaccio per lisare le cellule. <ol><li> Centrifugare la miscela a 10.000 xg per 10 min, separare il lisato dalla frazione pellet e misurare la concentrazione di proteine ​​totali di ciascun lisato utilizzando un acido bicinconinico (BCA) Kit test. </li></ol></li><li> Mescolare 40 ug di lisato con 4x SDS-PAGE tampone di caricamento (50 mM Tris-Cl, pH 6,8, 2% SDS, 10% glicerolo, ditiotreitolo 100 mM, 0,1% Bromophenol blu). <ol><li> Lessare i campioni per 10 minuti ed eseguire i campioni su un 12,5% di denaturazione SDS-PAGE ad una tensione costante di 200 V. </li></ol></li><li> Trasferire le proteine ​​separate su SDS-PAGE su una membrana di nitrocellulosa utilizzando la tecnica western blotting (ad una tensione costante di 90 V per 1 ora a freddo). <ol><li> Bloccare la membrana con 3% latte scremato in PBS 1x e quindi incubare la membrana con soluzioni di anticorpi primari diluiti (in questo caso coniglio anti-VDAC3, coniglio anti-γ-tubulina e topo anti-α-tubulina) per 1 ora a RT. </li><li> Dopo aver lavato la membrana tre volte (ogni 5 min di incubazione sotto agitando) usando tampone PBS-T (PBS 1x e 0,2% Tween-20), incubare la membrana in una miscela di infrarosso vicino (IR) colorante fluorescente coniugato anti-coniglio e colorante fluorescente IR coniugati anti-topo anticorpi secondari (diluito in PBS-T) per 1 ora. </li><li> Lavare la membrana per tre volte e quindi acquisire la membrana per analizzare le fasce con un f infrarossisistema di imaging luorescence adatto per il rilevamento immunoblot. </li></ol></li></ol>

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Cell Sci. 116, 4011-4019 (2003).</li><li>Korzeniewski, N., 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=Cullin+1+functions+as+a+centrosomal+suppressor+of+centriole+multiplication+by+regulating+polo-like+kinase+4+protein+levels.">Cullin 1 functions as a centrosomal suppressor of centriole multiplication by regulating polo-like kinase 4 protein levels.</a> Cancer Res. 69, 6668-6675 (2009).</li><li>Rohatgi, R., Milenkovic, L., Corcoran, R. B., Scott, 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=Hedgehog+signal+transduction+by+Smoothened:+pharmacologic+evidence+for+a+2-step+activation+process.">Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 3196-3201 (2009).</li><li>Majumder, S., Fisk, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VDAC3+and+Mps1+negatively+regulate+ciliogenesis.">VDAC3 and Mps1 negatively regulate ciliogenesis.</a> Cell Cycle. 12, 849-858 (2013).</li><li>Howell, B. J., Hoffman, D. B., Fang, G., Murray, A. W., Salmon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Mad2+dynamics+at+kinetochores,+along+spindle+fibers,+and+at+spindle+poles+in+living+cells.">Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells.</a> J. Cell Biol. 150, 1233-1250 (2000).</li><li>Meraldi, P., Nigg, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+cohesion+is+regulated+by+a+balance+of+kinase+and+phosphatase+activities.">Centrosome cohesion is regulated by a balance of kinase and phosphatase activities.</a> J. Cell Sci. 114, 3749-3757 (2001).</li><li>Zimmerman, W. C., Sillibourne, J., Rosa, J., Doxsey, 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=Mitosis-specific+anchoring+of+gamma+tubulin+complexes+by+pericentrin+controls+spindle+organization+and+mitotic+entry.">Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry.</a> Mol. Biol. 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J. 32, 1141-1154 (2013).</li><li>Lichtenstein, N., Geiger, B., Kam, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+cytoskeletal+organization+by+digital+fluorescent+microscopy.">Quantitative analysis of cytoskeletal organization by digital fluorescent microscopy.</a> Cytometry A. 54, 8-18 (2003).</li><li>Keller, L. 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=Molecular+architecture+of+the+centriole+proteome:+the+conserved+WD40+domain+protein+POC1+is+required+for+centriole+duplication+and+length+control.">Molecular architecture of the centriole proteome: the conserved WD40 domain protein POC1 is required for centriole duplication and length control.</a> Mol. Biol. Cell. 20, 1150-1166 (2009).</li><li>Venoux, 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=Poc1A+and+Poc1B+act+together+in+human+cells+to+ensure+centriole+integrity.">Poc1A and Poc1B act together in human cells to ensure centriole integrity.</a> J. Cell Sci. 126, 163-175 (2013).</li><li>Stevens, N. R., Roque, H., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DSas-6+and+Ana2+coassemble+into+tubules+to+promote+centriole+duplication+and+engagement.">DSas-6 and Ana2 coassemble into tubules to promote centriole duplication and engagement.</a> Dev. Cell. 19, 913-919 (2010).</li><li>Machiels, B. 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=Detailed+analysis+of+cell+cycle+kinetics+upon+proteasome+inhibition.">Detailed analysis of cell cycle kinetics upon proteasome inhibition.</a> Cytometry. 28, 243-252 (1997).</li><li>Yang, C. H., Kasbek, C., Majumder, S., Yusof, A. M., Fisk, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mps1+phosphorylation+sites+regulate+the+function+of+centrin+2+in+centriole+assembly.">Mps1 phosphorylation sites regulate the function of centrin 2 in centriole assembly.</a> Mol. Biol. Cell. 21, 4361-4372 (2010).</li><li>Tsou, M. 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The authors declare that they have no competing financial interests.

Microscopia quantitativa in biologia cellulare è comunemente associato con live-cell test di imaging come la Fluorescence Resonance Energy Transfer (FRET), Recupero della fluorescenza dopo photobleaching (FRAP), ecc, tuttavia, ci sono esempi di crescita di biologi cellulari di sviluppo diversi saggi microscopia quantitativi per fisso cellule negli ultimi anni 27,34-36. È importante sottolineare che, progressi nella comprensione della biologia centrosoma spesso richiede la comprensione della funzion...

Centrosomi costituiti da una coppia di centrioli circondate da materiale pericentriolar (PCM). Essendo i principali centri di organizzazione dei microtubuli (MTOC) in cellule di mammifero, centrosomi servono come i due poli di fusi mitotici nelle cellule in divisione, e quindi aiutano a mantenere l&#39;integrità genomica 1. Nelle cellule quiescenti (ad esempio, durante la fase G0), uno dei due centrioli del centrosoma, cioè centriolo madre, si trasforma in un corpo basale per assemblare il cilium primaria, un organello sensoriale sporge dalla superficie cellulare 2. Una volta che le cellule ri-entrare nel ciclo cellulare, cilia primaria sono smontati e ogni centriolo dirige il montaggio di un procentriole alla sua estremità prossimale che allunga progressivamente a formare una centriolo maturo 3. All&#39;inizio della fase S, una struttura di carro-like che fornisce la simmetria 9 volte al centriolo è formata sulla superficie di ciascun centriole esistente e diventerà la base di ogni procentriole. SAS6 tcappello è indispensabile per il montaggio centriolo viene reclutato per formare il fulcro della Cartwheel 4-6. Altre proteine ​​centriolar vengono poi assemblati sul cartwheel in un altamente regolamentato, prossimale a distale maniera 7. Dopo aver completato proprio centriolo duplicazione, cellule assemblare materiali pericentriolar supplementari per la costruzione di due centrosomi funzionali entro la fine della fase G2 8. Oltre ai componenti centriolar base 9-11, diverse altre proteine ​​tra cui chinasi, fosfatasi, accompagnatori, componenti del ponteggio, proteine ​​associate membrana e macchinari degrado sono associate a centrioli, corpi basali e PCM in momenti diversi del ciclo cellulare 12-16. Si è spesso osservato che i livelli centrosomica di molte proteine ​​sono temporalmente regolati da meccanismi di targeting centrosomica e / o degradazione del proteasoma in centrosomes. È importante sottolineare che le fluttuazioni del livello centrosomica di diverse proteine ​​come Plk4, Mps1, SAS6, e CP110 unt diversi punti del ciclo cellulare sembra essere essenziale per regolare centriole assemblaggio 5,17-22, e nel caso di Mps1 prevenire questa degradazione centrosomica porta alla formazione di centrioli eccesso 19. D&#39;altra parte, le frazioni centrosomica di diverse proteine ​​sono meno labile rispetto alle piscine citosolici. Per esempio siRNA-mediata down-regulation di Centrin 2 (Cetn2) ha comportato solo una riduzione moderata del livello di proteine ​​nelle centrioli nonostante grande riduzione dei suoi livelli di cellule intere 23. E &#39;quindi fondamentale per misurare le variazioni del livello delle proteine ​​centrosomica al centrosoma, piuttosto che misurare i livelli di proteine ​​intere cellule nel valutare le loro funzioni specifiche centrosoma. In questo studio abbiamo sviluppato un saggio con immunofluorescenza indiretta (IIF) per quantificare il livello relativo di una proteina a centrosomi. Questo test è stato sviluppato in particolare per analizzare cellule che sono da diversi campionie quindi non può essere ripreso contemporaneamente. Questi campioni possono essere le cellule che sono stati trattati con diversi reagenti (cioè, farmaco contro controllo), raccolti in diversi momenti (ad esempio, impulsi contro chase), o sono in diverse fasi del ciclo cellulare. Il principio di questo test consiste nel misurare l&#39;intensità di fluorescenza di fondo corretta corrispondente ad una proteina in una piccola regione e per normalizzare tale valore contro lo stesso per un&#39;altra proteina i cui livelli non variano nelle condizioni sperimentali adottate. Diversi studi in biologia centrosome hanno recentemente utilizzato varie tecniche di microscopia quantitative, in entrambe le cellule vive o fissi, per determinare la funzione specifica centrosoma di proteine ​​candidate 24-27. Simile a tali saggi, la tecnica attuale misura anche il fondo corretta intensità di fluorescenza della proteina. Tuttavia, l&#39;inclusione della normalizzazione usando uno standard interno in questo saggio sarebbe probabilmente offrire maggiorela precisione e la fiducia ad analizzare due campioni diversi che si trovano su due lamelle differenti. Inoltre, oltre ad esaminare il livello di proteine ​​a centrosomes, con piccole modifiche questo metodo può essere applicato ad un insieme eterogeneo di condizioni sperimentali o in altri siti cellulari. Qui, uniamo la nostra analisi di microscopia quantitativa con una strategia di pulse-chase BrdU per confrontare le cellule provenienti da diverse fasi del ciclo cellulare. Invece di utilizzare tecniche di arresto del ciclo cellulare normale e rilascio di studiare vari momenti del ciclo cellulare, le cellule in modo asincrono in crescita sono incubate con BrdU per etichettare le cellule in fase S, e le cellule marcate sono inseguiti per diverse volte (in genere 4-6 hr). La maggior parte delle cellule marcate saranno in fase S immediatamente dopo l&#39;impulso. La lunghezza della caccia è scelto in modo che dopo la caccia, cellule marcate saranno in ritardo S, G2 o mitosi, che può essere verificata da caratteristiche morfologiche tale posizione as- di centrosomi rispetto nuclei, distanza tra centrosomi, condensazione di cromosomi ecc Quindi, la lunghezza della caccia dipende dalla durata media di S, G2 e M di fase di un particolare tipo di cellula. Poiché questo approccio evita inibitori del ciclo cellulare come idrossiurea, afidicolina, nocodazole, ecc, permette una analisi più fisiologicamente rilevanti ciclo cellulare. Così, abbiamo dimostrato qui che la microscopia a fluorescenza quantitativa dosaggio soli, o in combinazione con BrdU test pulse-chase, è una tecnica semplice ma potente per misurare con precisione le relative variazioni del livello centrosomica di una proteina candidato durante un ciclo cellulare imperturbato. Abbiamo misurato il livello di centrosomica VDAC3, una proteina che abbiamo recentemente identificato in centrosomi, oltre ai mitocondri 16,28, con questi test. I risultati ottenuti qui verificare la nostra osservazione precedente che il pool di centrosomica VDAC3 è regolato da degrado, e varia anche in un dependen ciclo cellularet modo 16, inoltre, convalida l&#39;applicabilità di questo metodo.

<table border="0" cellpadding="0" cellspacing="0" width="576">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Name&#160;</td>
			<td style="width:105px;">Company&#160;</td>
			<td style="width:99px;">Catalog number</td>
			<td style="width:195px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:179px;">Fibronectin</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">F8141</td>
			<td style="width:195px;">Stock of 1 mg/ml in water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:179px;">DMSO</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">D2650</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:179px;">MG115</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">SCP0005</td>
			<td style="width:195px;">Stock of 10 mM in DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:179px;">BrdU</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">B5002</td>
			<td style="width:195px;">Stock of 10 mM in DMSO</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Anti-g-tubulin (mouse monoclonal, clone GTU 88)</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">T 6557</td>
			<td style="width:195px;">1:200 in IIF blocking buffer&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Anti-VDAC3 (rabbit polyclonal)&#160;</td>
			<td style="width:105px;">Aviva Systems Biology</td>
			<td style="width:99px;">ARP35180-P050</td>
			<td style="width:195px;">1:50 in IIF blocking buffer, 1:1000 in WB blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Anti-Sas6 (mouse monoclonal)&#160;</td>
			<td style="width:105px;">Santa cruz biotechnology</td>
			<td style="width:99px;">sc-81431</td>
			<td style="width:195px;">1:100 in IIF blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Anti-Cep135 (rabbit polyclonal)</td>
			<td style="width:105px;">Abcam</td>
			<td style="width:99px;">ab-75005</td>
			<td style="width:195px;">1:500 in IIF blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Anti-BrdU (rat monoclonal)&#160;</td>
			<td style="width:105px;">Abcam</td>
			<td style="width:99px;">ab6326</td>
			<td style="width:195px;">1:250 in IIF blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Alexa Fluor 350 Goat Anti-Rat IgG (H+L)</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21093</td>
			<td style="width:195px;">1:200 in IIF blocking buffer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) Antibody</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21202</td>
			<td style="width:195px;">1:1000 in IIF blocking buffer</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:179px;">Alexa Fluor 594 Donkey Anti-Mouse IgG (H+L) Antibody</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21203</td>
			<td style="width:195px;">1:1000 in IIF blocking buffer</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:179px;">Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21206</td>
			<td style="width:195px;">1:1000 in IIF blocking buffer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Alexa Fluor 594 Donkey Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21207</td>
			<td style="width:195px;">1:1000 in IIF blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Anti-g-tubulin (rabbit polyclonal)</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">T5192</td>
			<td style="width:195px;">1:1000 in WB blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Anti-a-tubulin (mouse monoclonal, DM1A)</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">T9026</td>
			<td style="width:195px;">1:20000 in WB blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Alexa Fluor 680 Donkey Anti-Rabbit IgG (H+L)</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A10043</td>
			<td style="width:195px;">1:10000 in WB blocking buffer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Mouse IgG (H&#38;L) Antibody IRDye800CW Conjugated</td>
			<td style="width:105px;">Rockland antibodies</td>
			<td style="width:99px;">610-731-002</td>
			<td style="width:195px;">1:10000 in WB blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">SlowFade Gold Antifade Reagent&#160;</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">S36936</td>
			<td style="width:195px;">Mounting media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Round coverslips 12CIR.-1</td>
			<td style="width:105px;">Fisherbrand</td>
			<td style="width:99px;">12-545-80</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Olympus IX-81 microscope</td>
			<td style="width:105px;">Olympus</td>
			<td style="width:99px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Retiga ExiFAST 1394 IR camera</td>
			<td style="width:105px;">QImaging&#160;</td>
			<td style="width:99px;">32-0082B-238</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">100X Plan Apo oil immersion objective</td>
			<td style="width:105px;">Olympus</td>
			<td style="width:99px;"></td>
			<td style="width:195px;">1.4 numerical aperture</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Slidebook software package</td>
			<td style="width:105px;">Intelligent Imaging Innovations</td>
			<td style="width:99px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Odyssey IR Imaging System</td>
			<td style="width:105px;">Li-cor Biosciences</td>
			<td style="width:99px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Bicinchoninic acid (BCA) assay</td>
			<td style="width:105px;">Thermo Scientific</td>
			<td align="right">23227</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-MNU2 Narrow UV cube</td>
			<td style="width:105px;">Olympus</td>
			<td>U-M622</td>
			<td>Filter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-MNU2 Narrow Blue cube</td>
			<td style="width:105px;">Olympus</td>
			<td>U-M643</td>
			<td>Filter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-MNU2 Narrow Green cube</td>
			<td style="width:105px;">Olympus</td>
			<td>U-M663</td>
			<td>Filter</td>
		</tr>
	</tbody>
</table>

quantitative immunofluorescence assay to measure the variation in protein levels at centrosomes

Centrosomi sono piccoli ma importanti organelli che servono come i poli di mitotico mandrino per mantenere l&#39;integrità genomica o assemblare cilia primario per agevolare le funzioni sensoriali nelle cellule. Il livello di una proteina può essere regolata in modo diverso a centrosomes rispetto ad altre posizioni .cellular, e la variazione nel livello centrosomica di diverse proteine ​​in punti diversi del ciclo cellulare sembra essere cruciale per la corretta regolazione del complessivo centriolo. Abbiamo sviluppato un microscopio a fluorescenza dosaggio quantitativo che misura variazioni relative del livello di una proteina a centrosomes in cellule fisse di diversi campioni, come in diverse fasi del ciclo cellulare o dopo trattamento con vari reagenti. Il principio di questo test consiste nel misurare l&#39;intensità di fluorescenza di fondo corretta corrispondente ad una proteina in una piccola regione, e normalizzare che misura contro lo stesso per un&#39;altra proteina che non varia con il c sperimentale presceltoondition. Utilizzando questo test, in combinazione con l&#39;impulso BrdU e la strategia caccia di studiare cicli cellulari imperturbati, abbiamo quantitativamente convalidato la nostra recente osservazione che il pool di centrosomica VDAC3 è regolata a centrosomi durante il ciclo cellulare, probabilmente dalla degradazione proteasoma-mediata specificamente a centrosomi.

I nostri studi recenti identificati romanzo localizzazione centrosomica e la funzione di VDAC3, uno dei porine mitocondriali 16,28. Immunostaining di diverse cellule di mammiferi, tra cui le cellule RPE1 utilizzando un anticorpo VDAC3 specifico ha mostrato prominente colorazione centrosomica e relativamente debole colorazione mitocondriale. Abbiamo anche dimostrato che VDAC3 centrosomica è preferenzialmente associata al centriolo madre, e la piscina centrosomica sia endogeno e ectopicamente espresso VDAC3 è...

Watch this Scientific Journal Video about Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes at JoVE.com

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Quantitative Immunofluorescence Assay misurare la variazione dei livelli di proteine ​​ad centrosomi

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

quantitative-immunofluorescence-assay-to-measure-variation-protein

Research

JoVE Journal

Biology

14.9K Views.  The Ohio State University. Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein’s fluorescence intensity to that of an appropriate internal standard. 

Video: Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Fotodecolorazione saggi (FRAP e FLIP) per misurare la dinamica delle proteine ​​della cromatina in Living cellule staminali embrionali

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Ricerca

Biologia

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Il MultiBac Protein Complex Piattaforma di produzione al EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

L&#39;analisi citofluorimetrica di bimolecular Complementation Fluorescenza: A High Throughput Metodo quantitativo per studiare l&#39;interazione proteina-proteina

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

I centrosomi Imaging in Fly Testicoli

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

La misurazione dei livelli Heme sintesi in cellule di mammifero

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro metilazione Assay per studiare proteine ​​Arginina Metilazione

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-base Espressione Proiezione di proteine ​​di membrana in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

In vitro misurare phosphatidylethanolamine metiltransferasi attività

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Uso di dispositivi microfluidici misurare la durata della vita e cellulare fenotipi in singole cellule di lievito in erba

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Avanzate tecniche di microscopia confocale per studiare le interazioni proteina-proteina e cinetica alle lesioni del DNA

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Quantitative Immunofluorescence Assay misurare la variazione dei livelli di proteine ​​ad centrosomi

Quantitative Immunofluorescence Assay misurare la variazione dei livelli di proteine ad centrosomi