Questo lavoro è sostenuto da fondi di avvio presso l&#39;Università di Louisville e CA135123 dal National Institutes of Health (per CH).

1. Cultura e transfezione cellulare <ol><li> COS-7 cellule vengono coltivate in terreno Eagle modificato di Dulbecco (DMEM) supplementato con 4,5 g / l di glucosio e 10% di siero bovino fetale (FBS). </li><li> Trasfezione plasmide è fatto con Lipofectamine secondo le istruzioni del produttore (Invitrogen). </li></ol> 2. Analisi al microscopio di fluorescenza Expression SNARE della superficie cellulare <ol><li> Il giorno prima della transfezione, 3 × 10 4 cellule COS-7 sono seminate su sterili 12-millimetri vetrini (Fisher Scientific) contenuti in piastre da 24 pozzetti. </li><li> Per v-celle in ciascun pozzetto, 0,25 microgrammi di plasmide che codifica tTA (pTet-Off, CLONTECH) è co-trasfettate con 0,25 mg di plasmidi che esprimono capovolto vampiri. </li><li> Per le cellule T in ogni pozzetto, 0,25 mg del plasmide codificante TRE-LacZ (PBI-G, CLONTECH) è co-trasfettate con 0,25 mg ciascuno dei plasmidi che esprimono capovolte SNAP-25 e sintaxine 1 o 4. </li><li> 24 ore dopo transfection, le cellule vengono fissate con 4% paraformaldeide in PBS + + (PBS supplementato con 0,1 g / l CaCl 2 e 0,1 g / l MgCl 2) per 10 min, e quindi bloccati nel 10% FBS in PBS + + per 30 min. </li><li> Le cellule sono incubate per 1,5 ore con gli anticorpi anti-Myc 9E10 anticorpi monoclonali (neat supernatante di coltura di ibridoma). </li><li> Dopo quattro lavaggi con PBS + +, le cellule vengono incubate per 1 ora con anticorpi secondari coniugati con FITC (Jackson ImmunoResearch Laboratories) alla diluizione di 1:500. </li><li> Dopo quattro lavaggi con PBS + +, le cellule marcate sono montati in oro antifade Prolungare reagente (Invitrogen). </li><li> Immagini confocali sono raccolti su un microscopio laser confocale Olympus scansione. Le immagini vengono elaborate con il software Adobe Photoshop. </li></ol> 3. Analisi FACS di espressione SNARE della superficie cellulare <ol><li> Il giorno prima della transfezione, 2 × 10 5 cellule COS-7 sono seminate in ciascun pozzetto di piastre da 6 pozzetti. </li><li> 24 ore dopo la tradinsfection con la capovolto SNARE, pTet-Off e pBI-G plasmidi, le cellule vengono fissate con 1% paraformaldeide in PBS + + per 15 min, e quindi bloccati nel 10% FBS in PBS + + per 15 min. </li><li> Le cellule sono incubate con gli anticorpi anti-Myc 9E10 anticorpi monoclonali per 60 min. </li><li> Dopo tre lavaggi con PBS + +, le cellule vengono marcate con anticorpi secondari coniugati con FITC (diluizione 1:200) per 45 min. </li><li> Dopo tre lavaggi con PBS + +, le cellule vengono raschiate le piastre con un raschietto cellulare. </li><li> 15.000 cellule vengono analizzate usando un citometro a flusso FACSCalibur (BD Biosciences). L&#39;intensità di fluorescenza media di ciascun campione è ottenuta utilizzando il software CellQuest Pro. </li></ol> 4. Enzimatica cellulare Assay Fusion <ol><li> Il giorno prima della transfezione, 1,2 x 10 6 cellule COS-7 sono seminate in ogni 100 mm piatto di coltura di tessuti, e 2 × 10 5 cellule COS-7 sono seminate in ciascun pozzetto di piastre da 6 pozzetti. </li><li> Per v-cellule, 0,5 - 5 mg ogni Flipped plasmide VAMP è cotransfected con 5 pg di pTet-Off nelle celle di ogni 100 mm piatto di coltura. Cellule di controllo sono co-trasfettate con il vettore vuoto pcDNA3.1 (+) e pTet-Off. Per impedire la N-glicosilazione di VAMPS 1, 4, 5, 7 e 8, tunicamicina (10 pg / ml) è incluso nel mezzo di coltura cellulare durante la trasfezione. </li><li> Per cellule T in ciascun pozzetto di piastre da 6 pozzetti, 1 ug di capovolte SNAP-25 e plasmide pBI-G vengono cotrasfettate con 0,5 pg di plasmide o capovolto syntaxin1 0,05 pg di plasmide syntaxin4 capovolte. </li><li> 24 ore dopo la trasfezione, le v-cellule sono staccate da piastre di coltura con l&#39;enzima senza dissociazione cellulare Buffer (Invitrogen). Cellule staccati sono contate con un emocitometro e risospese in tampone HEPES DMEM supplementato con 10% FBS, 6,7 ug / ml e 0,67 tunicamicina mM DTT. </li><li> 4,8 × 10 risospeso 5 V-cellule vengono aggiunti a ciascun pozzetto contenente già le cellule T. </li><li> Ratto dopo 6, 12 o 24 ore a 37 ° C in 5% CO 2 </sub&gt;, l&#39;espressione di β-galattosidasi è misurata utilizzando il β-galattosidasi Sistema saggio enzimatico con tampone di lisi Reporter secondo le istruzioni del produttore (Promega). Brevemente, le cellule vengono lavate due volte con PBS e lisate in tampone di lisi Reporter. I lisati cellulari sono mescolati con un volume uguale di tampone di saggio 2 ×. Come un controllo in bianco, il tampone di lisi Reporter viene miscelato con il tampone del saggio 2 ×. </li><li> Dopo 90 minuti, la reazione colorimetrica viene bloccata per aggiunta di carbonato di sodio 1 M. </li><li> Assorbanza a 420 nm viene misurata mediante spettrofotometro HITACHI 100-40. </li></ol>

<ol>
	<li>Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+intracellular+protein+transport.">Mechanisms of intracellular protein transport.</a> Nature. 372, 55-63 (1994).</li><li>Jahn, R., Lang, T., Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+fusion.">Membrane fusion.</a> Cell. 112, 519-533 (2003).</li><li>Bonifacino, J. S., Glick, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanisms+of+vesicle+budding+and+fusion.">The mechanisms of vesicle budding and fusion.</a> Cell. 116, 153-166 (2004).</li><li>Jahn, R., Scheller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNAREs--engines+for+membrane+fusion.">SNAREs--engines for membrane fusion.</a> Nature. 7, 631-643 (2006).</li><li>Weber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNAREpins:+minimal+machinery+for+membrane+fusion.">SNAREpins: minimal machinery for membrane fusion.</a> Cell. 92, 759-772 (1998).</li><li>Hu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+of+cells+by+flipped+SNAREs.">Fusion of cells by flipped SNAREs.</a> Science (New York, N.Y). 300, 1745-1749 (2003).</li><li>Hu, C., Hardee, D., Minnear, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+fusion+by+VAMP3+and+plasma+membrane+t-SNAREs.">Membrane fusion by VAMP3 and plasma membrane t-SNAREs.</a> Experimental cell research. 313, 3198-3209 (2007).</li><li>Hasan, N., Corbin, D., Hu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusogenic+Pairings+of+Vesicle-Associated+Membrane+Proteins+(VAMPs)+and+Plasma+Membrane+t-SNAREs+-+VAMP5+as+the+Exception.">Fusogenic Pairings of Vesicle-Associated Membrane Proteins (VAMPs) and Plasma Membrane t-SNAREs - VAMP5 as the Exception.</a> PloS one. 5, e14238 (2010).</li><li>Gossen, M., Bujard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+control+of+gene+expression+in+mammalian+cells+by+tetracycline-responsive+promoters.">Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.</a> Proceedings of the National Academy of Sciences of the United States of America. 89, 5547-5551 (1992).</li><li>Hanson, P. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle-associated+membrane+protein+4+is+implicated+in+trans-Golgi+network+vesicle+trafficking.">Vesicle-associated membrane protein 4 is implicated in trans-Golgi network vesicle trafficking.</a> Molecular biology of the cell. 10, 1957-1972 (1999).</li><li>Zeng, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+synaptobrevin/VAMP+homologous+protein+(VAMP5)+is+increased+during+in+vitro+myogenesis+and+present+in+the+plasma+membrane.">A novel synaptobrevin/VAMP homologous protein (VAMP5) is increased during in vitro myogenesis and present in the plasma membrane.</a> Molecular biology of the cell. 9, 2423-2437 (1998).</li><li>Galli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+tetanus+neurotoxin-insensitive+vesicle-associated+membrane+protein+in+SNARE+complexes+of+the+apical+plasma+membrane+of+epithelial+cells.">A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells.</a> Molecular biology of the cell. 9, 1437-1448 (1998).</li><li>Wong, S. 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Nessun conflitto di interessi dichiarati.

Il saggio originale fusione cellulare 6 determina SNARE mediati eventi di fusione cellulare mediante microscopia a fluorescenza. Qui si descrive un saggio innovativo che quantifica SNARE-mediati eventi di fusione di cellule di espressione attiva di β-galattosidasi e la misurazione spettrometrica. Questo test, si analizzano routinariamente 15-20-v e t-SNARE combinazioni in un singolo esperimento. Citometria a flusso per misurare SNARE espressione sulla superficie cellulare, abbiamo titolare i livelli di espre...

<table border="1"><tbody><tr><td> Nome del reagente e </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12800-017 </td></tr><tr><td> Cellule COS-7 </td><td> ATCC </td><td> CRL-1651 </td></tr><tr><td> tunicamicina </td><td> Sigma-Aldrich </td><td> T7765-5MG </td></tr><tr><td> pTet-Off </td><td> Clontech </td><td> K1620-A </td></tr><tr><td> PBI-G </td><td> Clontech </td><td> 6150-1 </td></tr><tr><td> lipofectamina </td><td> Invitrogen </td><td> 18324-012 </td></tr><tr><td> Enzima soluzione senza dissociazione cellulare </td><td> Invitrogen </td><td> 13151-014 </td></tr><tr><td> Coniglio anti-TET repressore anticorpo policlonale </td><td> Millipore </td><td> AB3541 </td></tr><tr><td> FITC-coniugato asino anti-IgG di topo (H + L) </td><td> Jackson ImmunoResearch </td><td> 715-095-150 </td></tr><tr><td> Microscopio a scansione laser confocale </td><td> Olimpo </td><td> FV1000 </td></tr><tr><td> FACSCalibur citofluorimetro </td><td> BD Biosciences </td><td></td></tr><tr><td> β-galattosidasi Assay sistema enzimatico con il tampone di lisi Reporter </td><td> Promega </td><td> E2000 </td></tr><tr><td> Modello 100-40 UV-VIS </td><td> Hitachi </td><td> C740843 </td></tr></tbody></table>

analysis of snare-mediated membrane fusion using an enzymatic cell fusion assay

Le interazioni di SNARE (s oluble N-ethylmaleimide sensibile al fattore di una proteina recettore ttachment ri) proteine ​​sulla vescicole (v-SNARE) e membrane bersaglio (t-SNARE) catalizzano la fusione della vescicola intracellulare 1-4. Saggi di ricostituzione, sono essenziali per la dissezione del meccanismo e la regolazione di SNARE-mediata fusione della membrana 5. In un saggio di fusione cellulare 6,7, proteine ​​SNARE sono espresse ectopicamente alla superficie cellulare. Questi &quot;girato&quot; SNARE unità proteine ​​fusione cellula-cellula, dimostrando che SNARE sono sufficienti per fondere le membrane cellulari. Poiché il saggio fusione cellulare si basa su analisi microscopica, è meno efficace quando viene utilizzato per analizzare multiple e v-t-SNARE interazioni quantitativamente. Qui si descrive un nuovo saggio 8 che quantifica SNARE-mediati eventi di fusione di cellule dall&#39;espressione attiva di β-galattosidasi. Duecomponenti del sistema Tet-Off genica 9 sono utilizzati come visualizzatore sistema: la tetraciclina controllato transattivatore (tTA) ed un plasmide reporter che codifica il gene lacZ sotto il controllo della tetraciclina-risposta elemento (TRE-LacZ). Abbiamo transfect tTA in cellule COS-7 che esprimono capovolte v-SNARE proteine ​​sulla superficie cellulare (V-cellule) e trasfezione TRE-LacZ in cellule COS-7 che esprimono capovolte t-SNARE proteine ​​sulla superficie cellulare (cellule T) . SNARE-dipendente fusione delle V-e cellule T determina il legame di tTA al TRE, l&#39;attivazione trascrizionale di LacZ e espressione di β-galattosidasi. L&#39;attività di β-galattosidasi è quantificato mediante un metodo colorimetrico mediante assorbimento a 420 nm. Le vescicole associate proteine ​​di membrana (vampiri) sono v-SNARE che risiedono in vari post-Golgi compartimenti vescicolari 10-15. Esprimendo VAMPS 1, 3, 4, 5, 7 e 8 allo stesso livello, si confronta la fusione della membranaattività che utilizzano il enzimatica fusione cellulare. Basata sulla misura spettrometrica, questo saggio offre un approccio per l&#39;analisi quantitativa SNARE-mediata fusione della membrana e di alta produttività studi.

Per sviluppare un saggio quantitativo fusione cellulare, approfittiamo del forte attivazione trascrizionale dal legame di tTA a TRE. In assenza di tTA, trascrizione del gene LacZ in TRE-LacZ è silenzioso. Quando tTA è presente, si lega al TRE e attiva la trascrizione di LacZ. Figura 1 mostra un diagramma di flusso del saggio enzimatico fusione cellulare. tTA viene transfettato in v cellule che esprimono v-SNARE proteine ​​sulla superficie cellulare, e TRE-LacZ v...

Watch this Scientific Journal Video about Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay at JoVE.com

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

Abbiamo sviluppato un saggio fusione cellulare che quantifica SNARE mediati eventi di fusione della membrana mediante espressione di β-attivato galattosidasi.

Analisi delle SNARE-mediata fusione della membrana L&#39;utilizzo di un test enzimatico fusione cellulare

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

analysis-snare-mediated-membrane-fusion-using-an-enzymatic-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.9K Views.  University of Louisville School of Medicine. Abbiamo sviluppato un saggio fusione cellulare che quantifica SNARE mediati eventi di fusione della membrana mediante espressione di β-attivato galattosidasi.

Video: Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

Un'analisi di spettroscopia di fluorescenza fluttuazione delle interazioni proteina-proteina a contatti cellula-cellula

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Ricerca

Biochimica

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Ricostituzione assistita da detergenti della Drosophila ricombinante Atlastin in Liposomi per i saggi che mescolano i lipidi

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

Una piattaforma di membrana modello per la ricostituzione delle dinamiche della membrana mitocondriale

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Metodo per la misurazione di Cinetica fusione virale a livello di singola particella

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Biologia

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Elettrosaldabili cellula visualizzate con microscopia a fluorescenza

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizzando metodologie combinati per definire il ruolo della membrana plasmatica di consegna Durante Axon ramificazione e neuronale morfogenesi

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Neuroscienze

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

Fusion SNARE-mediata del singolo proteoliposomi con bilayers Tethered supportati in una cella di flusso Microfluidic monitorati da polarizzata microscopia TIRF

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Visualizing intracellulare rullante traffico da fluorescenza Lifetime Imaging microscopia

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Immunologia e infezioni

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Mitochondrial fusion was measured by tracking the equilibration of photoconverted matrix-targeted GFP across the mitochondrial network over time. Thus far, only one cell could be subjected to an hour long kinetic analysis at a time. We present a method that simultaneously measures multiple cells, thereby speeding up the data collection process.

Una più veloce, ad alta risoluzione, MTPA-GFP-based test Fusion mitocondriale Acquisizione dati cinetici di più celle in parallelo con microscopia confocale

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated &#937;-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C &#948; (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

Microscopia confocale per misurare tre modalità di dinamica dei pori di fusione nelle cellule cromaffini surrenali

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...

Analisi delle SNARE-mediata fusione della membrana L'utilizzo di un test enzimatico fusione cellulare

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