Gli autori ringraziano René Scriwanek per l&#39;assistenza alla preparazione della figura. FR è sostenuto da ECHO (700.59.003), ALW programma aperto (821.02.017 e 822.02.014), la cooperazione DFG-NWO (DN82-303) e ZonMw VICI (016.130.606) sovvenzioni.

1. Spheroplast Preparazione <ol><li> Incubare il lievito notte a 30 ° C in 10 ml di mezzo appropriato determinato dal disegno dell&#39;esperimento. </li><li> Il giorno dopo, misurare la densità ottica della coltura a 600 nm (OD 600) usando un fotometro, Diluire le cellule nello stesso terreno di coltura ad una OD 600 di 0,2-0,4 e farle crescere ad una fase di crescita esponenziale meno che la progettazione del esperimento richiede una condizione diversa. Le cellule sono in fase esponenziale quando la coltura ha un diametro esterno 600 di 1-2. </li><li> Raccogliere 10 OD 600 equivalenti di cellule mediante centrifugazione a 3500 xg per 5 minuti in un tubo da 50 ml. Dopo la centrifugazione, scartare il surnatante. </li><li> Risospendere il pellet di cellule in 5 ml di tubi 100 mM (pH 9,6), ditiotreitolo 10 mM e incubate a 30 ° C per 10 min. </li><li> Raccogliere nuovamente le cellule per centrifugazione a 3.500 xg per 5 min. Scartare il surnatante. </li><li> Risospendere le cellule in 5 ml di terreno (determinato dal disegno dell&#39;esperimento) contenente 1 M sorbitolo e 5 mg di enzima litico, e incubare la miscela a 30 ° C con delicata agitazione per 30 min. </li><li> Centrifugare la sospensione cellulare a 300 xg per 5 minuti per raccogliere la frazione di pellet, che corrisponde alle sferoplasti. Scartare il surnatante. </li><li> Risospendere le sferoplasti in 960 ml di ghiaccio freddo supporti (medio determinato dal disegno dell&#39;esperimento) contenente 1 M sorbitolo e trasferire il composto in una ghiacciata 2 ml provetta. </li></ol> 2. Nanogold assorbimento <ol><li> Usando una pipetta, mescolare delicatamente il spheroplast con 4 nmol di particelle cariche positivamente nanogold risospese in 40 ml di acqua. Il volume finale della miscela deve essere di 1 ml. </li><li> Posizionare la sospensione cellulare ottenuta in ghiaccio per 15 min. </li><li> Trasferire la sospensione a temperatura ambiente ed incubare per il tempo necessario a consentire nanogold internalizzazione via endocytosis. NOTA: A 5 min assorbimento sarà principalmente portare l&#39;etichettatura di vescicole e dei primi comparti endosomiali, mentre un 15 min assorbimento permetterà di contrassegnare l&#39;intero sistema endosomiale (precoce e endosomi tardivi). Tempi di incubazione più lungo (più di 30 min), consentiranno inoltre di etichettare vacuolo. NOTA: esperimenti di etichettatura Pulse-chase non possono essere eseguite con questo metodo. Pertanto, quando l&#39;etichettatura per l&#39;analisi di LE, nanogold anche essere trovate in PM e INEE. </li></ol> 3. Fissazione e sezionamento <ol><li> Interrompere l&#39;assorbimento nanogold con l&#39;aggiunta di 1 ml di doppio fissativo forza [4% paraformaldeide (PFA), 0,4% glutaraldeide (GA) in 0.1 M tampone PHEM (20 TUBI mm, 50 HEPES mM, pH 6,9, 20 mM EGTA, MgCl 4 mM 2)] contenente 1 M sorbitolo alla sospensione cellulare. Mantenuto il tubo a temperatura ambiente. </li><li> Capovolgere delicatamente manualmente le provette micocentrifuge più volte durante 30 minuti (questo permetterà di mantenere le sferoplasti insospensione) e centrifugare due volte a 1700 xg per 25 sec. </li><li> Sostituire il fissativo scartando il surnatante e con l&#39;aggiunta di 1 ml di fissativo fresco forza standard (2% PFA, 0,2% GA in 0.1 M tampone PHEM) contenente 1 M sorbitolo e incubare per 2 ore a temperatura ambiente su una ruota lenta rotazione della camera. </li><li> Elaborare le cellule per crio-sezionamento come descritto in precedenza 6. NOTA: Per le procedure di valorizzazione nanogold assorbimento e d&#39;argento, il trattamento con acido periodico descritto nel protocollo indicato è di non essere effettuati. Il trattamento con acido periodico è permeabilize meglio la parete cellulare 26 ma questa struttura è stata eliminata durante la generazione di sferoplasti. </li><li> Tagliare 50 criosezioni sottili nm a -120 ° C con la lama di diamante a secco utilizzando un UCT ultramicrotomo come descritto in precedenza 8 e metterli su carbone Formvar rivestito 50 maglie griglie di nichel. </li></ol> 4. Miglioramento argento per Nanogold particelle Visualizzazione NOTA: La procedura per preparare le miscele di reazione è fondamentalmente quello indicato dal costruttore. Adeguamenti pratici che utilizzano questo protocollo, rende la procedura di valorizzazione argento efficace e affidabile su criosezioni. <ol><li> Rimuovere il kit di potenziamento argento dal freezer e scongelare in un incubatore a 37 ° C oa bagnomaria. Quando scongelati, posizionare il kit in un incubatore a 24 ° C collocato in una stanza buia fino al momento dell&#39;uso (vedi 4.7). </li><li> Posizionare la piastra di riscaldamento in camera oscura e riscaldarlo alla temperatura finale di 24 ° C. </li><li> Coprire la parte superiore della piastra di riscaldamento con la protezione di superficie, lato lucido, e fissarlo con un nastro. </li><li> Posizionare il Parafilm sul Benchkote e segnare i bordi con un pennarello nero per essere in grado di vedere i bordi parafilm nel buio. </li><li> Nastro un termometro sulla parte superiore della Benchkote per monitorare la temperatura della piastra riscaldante. Gradualmente regolare la temperatura se non 24 ° C. </li><li> PlaCE Il tubo da 50 ml con acqua bidistillata e il kit di miglioramento argento all&#39;interno. </li><li> Riempire le piccole piastre di Petri con acqua bidistillata, pre-riscaldato a 37 ° C. Posizionare le griglie in acqua, provino lato negativo, per 30 min. </li><li> Ripetere questo passo 4.7 ancora una volta. </li><li> Trasferire le griglie nel box di stoccaggio e portarli alla camera oscura. </li><li> Risciacquare nuovamente le griglie facendoli passare (con il lato campione giù) su alcune gocce di acqua bidistillata a 24 ° C immessi sul parafilm che è stato fissato sulla piastra di riscaldamento. </li><li> Assicurarsi che 9 supplementare doppio gocce d&#39;acqua distillata sono pronti sul Parafilm, per il risciacquo dopo la reazione valorizzazione argento. </li><li> Spegnere la luce e assicurarsi che tutte le luci siano spente. Accendere la luce rossa. </li><li> Estrarre le soluzioni A e B del kit potenziamento argento dal termostato 24 ° C. </li><li> Mettere primi 6 gocce di soluzione A e poi 6 gocce di soluzione B in un 1,5 ml provetta e mescolare bene con una pipetta Pasteur di vetro evitando di fare le bolle. La soluzione è abbastanza spessa e deve essere miscelato manualmente con una pipetta prima di finalmente vortex brevemente. </li><li> Mettere le soluzioni A e B di nuovo in incubatrice 24 ° C ed estrarre la soluzione C. </li><li> Aggiungere 6 gocce della soluzione C al mix A / B. Poi mescolare ancora prima con una pipetta Pasteur e poi nel vortex, evitare di fare bolle. </li><li> Utilizzare immediatamente la miscela finale inserendo (~ 20 l / drop) gocce sul Parafilm. </li><li> Con un pulito, pinzette anti-magnetico, posizionare le griglie sulla miscela (ad es., Argento migliorando reazione) da 6 a 15 minuti a seconda della valorizzazione (ie. Dimensione delle particelle d&#39;oro) voluto da ottenere. </li><li> Rimuovere le griglie dalla miscela e trasferirli sulla superficie dell&#39;acqua bidistillata scende a 24 ° C per lavaggi. In primo luogo, utilizzare in rapida successione 6 gocce e poi 3 gocce con 7 min tempo di incubazione per goccia, Prima di accendere le luci. </li><li> Procedere con il passaggio successivo, sia l&#39;etichettatura immuno-oro o marcatura di membrana 27, al fine di visualizzare il risultato di un&#39;inchiesta EM. </li></ol>

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The authors have nothing to disclose.

Immuno-microscopia elettronica è una tecnica che permette di combinare localizzazione di proteine ​​con la risoluzione ultrastrutturale dei vettori e organelli dove queste proteine ​​risiedono. Ciò è particolarmente importante quando si studia il sistema lievito endosomiale perché i suoi comparti appaiono come strutture puntuate in microscopia a fluorescenza. È pertanto difficile distinguerle. Per questo motivo l&#39;uso di una sonda rilevabile da EM e immettendo il percorso endocitosi in modo dipendente da...

Il sistema endosomal è un importante apparato di smistamento membrana che svolge molteplici ruoli cellulari cruciali, tra cui il traffico di recettori smistamento enzima lisosomiale e il riciclaggio di membrana plasmatica (PM) recettori 1,2. Endosomi sono divisi in tre diversi comparti, vale a dire. gli endosomi precoci (EE), alla fine degli endosomi (LE) e le endosomi di riciclo. Questa classificazione si basa sul tempo impiegato materiale endocitosi raggiungerli, sulle proteine ​​marker specifiche e sulla loro morfologia. Membrane, ovvero proteine ​​e lipidi bistrati, interiorizzate dal PM possono sia essere consegnati ai lisosomi via endosomi per la degradazione o essere riciclati. Le membrane sono trasportati endosomi dal Golgi e similmente, sia continuare ad lisosomi o essere recuperate indietro ai Golgi. Inoltre, le proteine ​​possono essere filtrate in vescicole luminali erba verso l&#39;interno dalla membrana endosomal limitante, un processo che porta alla formazione diuna sottocategoria di LE, i corpi multivescicolari. Il sistema endosomal lievito è relativamente meno complessa di quella delle cellule eucariotiche superiori. Endosomi lievito sono suddivisi in EE e LE. In contrasto con cellule di mammifero non contengono endosomi di riciclo ma anche organelli lisosomi legati tessuto-specifici. Di conseguenza, essi hanno una rete meno complessa di endosomal rotte del narcotraffico 3,4. Pertanto lievito ha rappresentato e rappresenta un sistema sperimentale vantaggiosa per studiare alcuni dei principi traffico membrana sottostante nel sistema endosomal. Questo vantaggio è accentuato dal fatto che numerosi geni coinvolti nelle vie endosomali sono stati inizialmente isolato con schermi genetici nel lievito 5. Mentre il sistema endosomiale lievito wild type e cellule mutanti è stata ampiamente studiata utilizzando approcci biochimici e di microscopia a fluorescenza, la sua indagine a livello ultrastrutturale è stato solominima. Analisi morfologiche sono particolarmente rilevanti nel lievito perché la maggior parte degli organelli endosomiali vengono rilevati come strutture puntuate mediante microscopia a fluorescenza, che rendono difficile la loro identificazione univoca 6. Purtroppo solo un numero limitato di antisieri riconoscere proteine ​​marker lievito endosomali sta lavorando in immuno-microscopia elettronica (IEM) preparazioni 7-10. Per alcune proteine, questo problema è stato aggirato dalla codifica endogeno del gene di interesse e l&#39;impiego di un anticorpo che riconosce il tag di rilevarla 7,11,12. Spesso, tuttavia, le proteine ​​sono rilevabili da IEM causa dei loro livelli di espressione bassi. La loro sovraespressione non è una soluzione, perché questo approccio può indurre mis-localizzazioni e / o alterazioni nei organelli morfologia / funzioni. Così l&#39;etichettatura dei compartimenti endocitici con una sonda rilevabile mediante microscopia elettronica EM è un&#39;opzione efficace. Questa è una soluzione ottimale soprattutto se il prOBE sta entrando nel percorso endocitosi in modo dipendente dal tempo, che permette di conoscere quando segnerà un organello specifico 6. L&#39;assorbimento di nanogold carica positiva da sferoplasti lievito (vale a dire. Lievito in cui la parete cellulare è stato rimosso enzimaticamente) è stato utilizzato con successo per identificare la endosomiale lievito scomparti 10. Queste particelle si legano fortemente ai lipidi carichi negativamente che compongono le membrane biologiche. Così i soci nanogold cariche positivamente con il PM, penetra nella cellula per endocitosi e passa attraverso l&#39;EE e LE prima di raggiungere il vacuolo. Queste piccole particelle d&#39;oro, tuttavia, non hanno dimensioni adeguate per essere visto da EM. Per renderle visibili, le loro dimensioni possono essere allargata mediante reazioni chimiche che portano alla deposizione di argento o oro intorno alla sonda oro 13-15. Abbiamo sviluppato e applicato con successo un approccio IEM basato sul metodo Tokuyasu effettuare subcellularelocalizzazione studia 8,16. Questo metodo consente di eseguire etichettatura immunogold su preparazioni di lievito con una risoluzione eccellente della morfologia 8,17-24. Abbiamo anche stabilito una procedura che combina questo protocollo IEM con l&#39;etichettatura nanogold del sistema lievito endosomale compartimenti 6. Usando questo approccio abbiamo morfologicamente caratterizzato diverse sottoclassi di endosomi e ultrastrutturalmente esaminato mutanti con un traffico endosomiale difetto 6,25. Inoltre, abbiamo dimostrato che questa etichettatura nanogold può essere combinato con etichettature immunogold fornendo la possibilità di esplorare la distribuzione di una proteina di interesse sulle diverse sottopopolazioni endosomi. Qui vi presentiamo come è praticamente eseguito l&#39;etichettatura del sistema lievito endosomiale con nanogold carica positiva.

<table><tbody><tr><td>PIPES (piperazin-1,4-bis (2-ethansulfensaure)</td><td>Merck, Darmstadt, Germany</td><td>1,102,200,250</td><td></td></tr><tr><td>HEPES</td><td>Merck, Darmstadt, Germany</td><td>391,340</td><td></td></tr><tr><td>EGTA</td><td>Sigma, St louis, MO</td><td>E4378</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck, Darmstadt, Germany</td><td>1,058,330,250</td><td></td></tr><tr><td>DL-Dithiothreitol</td><td>Sigma, St louis, MO</td><td>D0632</td><td></td></tr><tr><td>Sorbitol</td><td>Sigma, St louis, MO</td><td>S1876</td><td></td></tr><tr><td>Positively charged Nanogold</td><td>Nanoprobes, Yaphank, NY</td><td>2022</td><td>store at -20 &amp;deg;C</td></tr><tr><td>HQ-silver&amp;trade; enhancement kit</td><td>Nanoprobes, Yaphank, NY</td><td>2012</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma, St louis, MO</td><td>441244</td><td></td></tr><tr><td>Glutaraldehyde 8% EM grade</td><td>Polyscience, Inc., Warrington, PA</td><td>216</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Lytic enzyme</td><td>MP Biomedicals, Santa Ana, CA</td><td>153526</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Parafilm M</td><td>Sigma, St louis, MO</td><td>P7793</td><td></td></tr><tr><td>50 meshes nickel grids&amp;nbsp;</td><td>Agar Scientific, Stansted, United Kingdom</td><td>G209N</td><td></td></tr><tr><td>Cryo-immuno diamond knife 35&amp;deg;</td><td>Diatome AG, Biel, Switzerland</td><td>N.A.</td><td></td></tr><tr><td>UCT ultramicrotome&amp;nbsp;</td><td>Leica, Solms, Germany</td><td>N.A.</td><td></td></tr><tr><td>Formvar 15/95 resine</td><td>Electron Microscopy Sciences, Hatfield, PA</td><td>15800</td><td></td></tr><tr><td>50 meshes nickel grids&amp;nbsp;</td><td>Agar Scientific, Stansted, United Kingdom</td><td>AGG2050N</td><td></td></tr><tr><td>Parafilm</td><td>Sigma, St louis, MO</td><td>P7793</td><td></td></tr><tr><td>Grids storage box</td><td>Leica, Solms, Germany</td><td>162-50</td><td></td></tr><tr><td>Falcon 100 mm x 15 mm Petri dish</td><td>Corning, Corning, NY</td><td>351029</td><td></td></tr><tr><td>Pasteur capillary pipettes 150 mm</td><td>Van Bruggen Glas, Rotterdam, The Netherlands</td><td>10216234</td><td></td></tr><tr><td>1.5 ml microcentrifuge&amp;nbsp;</td><td>Sarstedt, N&amp;uuml;mbrecht, Germany</td><td>72690001</td><td></td></tr><tr><td>50 ml tube</td><td>Bio-one, Alphen aan den Rijn, The Netherlands</td><td>210296</td><td></td></tr><tr><td>Benchkote surface protector</td><td>Whatman, Maidstone, United Kingdom&amp;nbsp;</td><td>1159201</td><td></td></tr></tbody></table>

nanogold labeling of the yeast endosomal system for ultrastructural analyses

Endosomi sono uno dei principali membrana ordinamento posti di blocco nelle cellule eucariotiche e regolano il riciclaggio o la distruzione delle proteine ​​per lo più dalla membrana plasmatica e il Golgi. Di conseguenza il sistema endosomal svolge un ruolo centrale nel mantenimento dell&#39;omeostasi cellulare, e mutazioni in geni appartenenti a questa rete di organelli interconnessi da trasporto vescicolare, causano gravi patologie tra cui il cancro e disturbi neurobiologici. E &#39;quindi di primaria importanza per comprendere i meccanismi alla base della biogenesi e l&#39;organizzazione del sistema endosomal. Il lievito Saccharomyces cerevisiae è stato fondamentale in questo compito. Per etichettare e analizzare a livello ultrastrutturale sistema endosomal di questo organismo modello specifico, presentiamo qui un protocollo dettagliato per la nanogold assorbimento carica positiva da sferoplasti seguite dalla visualizzazione di queste particelle attraverso una reazione valorizzazione argento. Questo metodo è anche un prezioso troppol per l&#39;esame morfologico di mutanti con difetti nel traffico endosomal. Inoltre, non è solo applicabile per esami ultrastrutturali ma può anche essere combinato con etichettature immunogold delle indagini localizzazione della proteina.

Secondo il protocollo presentato, la morfologia del sistema lievito endosome può essere accesso da EM trasmissione. Figura 1 mostra diversi tipi di compartimenti endosomiali che sono stati raggiunti e quindi etichettati con nanogold. Il nanogold-argento avanzato può essere chiaramente visto come elettroni particelle dense. Le impostazioni ottimali stabiliti per la reazione valorizzazione argento permette avente particelle di oro in una gamma omogenea dimensioni tra 5 e 15 nm, che non interferisce con ...

Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Lievito di birra, Saccharomyces cerevisiae, è stata un organismo modello chiave per identificare e studiare geni che regolano la biogenesi e le funzioni del sistema endosomal. Qui vi presentiamo un protocollo dettagliato per l&#39;etichettatura specifico dei comparti endosomiali per gli studi ultrastrutturali.

Nanogold Etichettatura del Sistema lievito endosomiale per le analisi ultrastrutturali

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

nanogold-labeling-yeast-endosomal-system-for-ultrastructural

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.2K Views.  University Medical Center Utrecht.  Lievito di birra, Saccharomyces cerevisiae, è stata un organismo modello chiave per identificare e studiare geni che regolano la biogenesi e le funzioni del sistema endosomal. Qui vi presentiamo un protocollo dettagliato per l'etichettatura specifico dei comparti endosomiali per gli studi ultrastrutturali. 

Video: Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Un high-throughput metodo per studiare la morfologia globale organello in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Ricerca

Biologia

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Post-embedding Etichettatura immuno di proteine ​​sinaptiche in Culture Slice ippocampali

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Neuroscienze

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Applicazione di nanoparticelle fluorescenti per studiare rimodellamento del sistema Endo-lisosomiale da batteri intracellulari

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Immunologia e infezioni

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Etichettatura e visualizzazione dei prodotti di espressione del genoma mitocondriale nel lievito di Baker Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Biochimica

Visualizing Yeast Organelles with Fluorescent Protein Markers

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

Here we describe the use of a set of fluorescent protein-based organelle markers in live-cell imaging of the budding yeast, Saccharomyces cerevisiae.

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have ...

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 ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

Le domande di pHluorin per quantitativa, Cinetica e high-throughput analisi di endocitosi in germogliamento lievito

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize macromolecular complexes at very high resolution. However, to get as close as possible to the native state, perfect sample preservation is required. Conventional electron microscopy (EM) fixation with aldehydes, for instance, does not provide good ultrastructural preservation. The slow penetration of fixatives induces cell reorganization and loss of various cell components. Therefore, conventional EM fixation does not allow for an instantaneous stabilization and preservation of structures and antigenicity. The best choice for examining intracellular events is to use cryofixation followed by the freeze-substitution fixation method that keeps cells in their native state. High-pressure freezing/freeze-substitution, which preserves the integrity of cellular ultrastructure, is the most commonly used method, but requires expensive equipment. Here, an easy-to-use and low-cost freeze fixation method followed by freeze-substitution for suspension cell cultures is presented.

This manuscript describes an easy-to-use and low-cost cryofixation method for visualizing suspension cells by transmission electron microscopy.

Plunge congelamento: uno strumento per l&#39;ultrastrutturali e Immunolocalizzazione Studi di cellule in sospensione in microscopia elettronica a trasmissione

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

Microscopia 4D del lievito

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Esame della dinamica nucleare del lievito di fissione mitotico e meiotico da fluorescenza microscopia a celle vive

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Nanogold Etichettatura del Sistema lievito endosomiale per le analisi ultrastrutturali

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Capitoli in questo video

Un high-throughput metodo per studiare la morfologia globale organello in S. cerevisiae

Post-embedding Etichettatura immuno di proteine sinaptiche in Culture Slice ippocampali

Applicazione di nanoparticelle fluorescenti per studiare rimodellamento del sistema Endo-lisosomiale da batteri intracellulari

Etichettatura e visualizzazione dei prodotti di espressione del genoma mitocondriale nel lievito di Baker Saccharomyces cerevisiae

Visualizing Yeast Organelles with Fluorescent Protein Markers

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Esame della dinamica nucleare del lievito di fissione mitotico e meiotico da fluorescenza microscopia a celle vive

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Un high-throughput metodo per studiare la morfologia globale organello in S. cerevisiae

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Applicazione di nanoparticelle fluorescenti per studiare rimodellamento del sistema Endo-lisosomiale da batteri intracellulari

Etichettatura e visualizzazione dei prodotti di espressione del genoma mitocondriale nel lievito di Baker Saccharomyces cerevisiae

Visualizing Yeast Organelles with Fluorescent Protein Markers

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

Le domande di pHluorin per quantitativa, Cinetica e high-throughput analisi di endocitosi in germogliamento lievito

Plunge congelamento: uno strumento per l'ultrastrutturali e Immunolocalizzazione Studi di cellule in sospensione in microscopia elettronica a trasmissione

Microscopia 4D del lievito

Esame della dinamica nucleare del lievito di fissione mitotico e meiotico da fluorescenza microscopia a celle vive

Post-embedding Etichettatura immuno di proteine sinaptiche in Culture Slice ippocampali