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>

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	<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. I., Heuser, J. E., Jahn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotransmitter+release+-+four+years+of+SNARE+complexes.">Neurotransmitter release - four years of SNARE complexes.</a> Current opinion in neurobiology. 7, 310-315 (1997).</li><li>McMahon, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellubrevin+is+a+ubiquitous+tetanus-toxin+substrate+homologous+to+a+putative+synaptic+vesicle+fusion+protein.+[see+comments.">Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein. [see comments.</a> Nature. 364, 346-349 (1993).</li><li>Steegmaier, M., Klumperman, J., Foletti, D. L., Yoo, J. S., 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=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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endobrevin,+a+novel+synaptobrevin/VAMP-like+protein+preferentially+associated+with+the+early+endosome.">Endobrevin, a novel synaptobrevin/VAMP-like protein preferentially associated with the early endosome.</a> Molecular biology of the cell. 9, 1549-1563 (1998).</li></ol>

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

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

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 ...

Ricerca

Biologia

Anterior Cervical Discectomy and Fusion in the Ovine Model

Anterior cervical discectomy and fusion (ACDF) is the most common surgical operation for cervical radiculopathy and/or myelopathy in patients who have failed conservative treatment1,5. Since the operation was first described by Cloward2 and Smith and Robinson6 in 1958, a variety refinements in technique, graft material and implants have been made3. In particular, there is a need for safe osteoinductive agents that could benefit selected patients. The ovine model has been shown to have anatomical, biomechanical, bone density and radiological properties that are similar to the human counterpart, the most similar level being C3/44. It is therefore an ideal model in which preclinical studies can be performed. In particular this methodology may be useful to researchers interested in evaluating different devices and biologics, including stem cells, for potential application in human spinal surgery.

This video demonstrates the technique of anterior cervical discectomy and fusion in the ovine model.

Discectomia cervicale anteriore e fusione nel modello ovina

Anterior cervical discectomy and fusion (ACDF) is the most common surgical operation for cervical radiculopathy and/or myelopathy in patients who have ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

Etichettatura fluorescente di COS-7 Esprimendo SNAP-tag proteine ​​di fusione per l&#39;imaging cellulare dal vivo

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

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 ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Espressione di proteine di fusione fluorescenti in macrofagi e cellule dendritiche derivate da midollo osseo Murine

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

Analisi di assorbimento di colesterolo LDL utilizzando Live Cell Imaging analisi con cella di monitoraggio dello stato

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Effetto di proteine fluorescenti su Fusion partner utilizzando saggi di tossicità di Polyglutamine in lievito

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

Sfruttando Live Imaging ai Nuclei di pista durante il differenziamento dei mioblasti e Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing&#160;strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment&#39;s purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

We describe here a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in Nicotiana benthamiana. This protocol can be extended to purification and visualization of numerous proteins that utilize column chromatography. Moreover, the protocol is adaptable to the in-person and virtual college teaching laboratory, providing project-based exploration.

Produzione di proteine di fusione IgG espresse transitoriamente in Nicotiana benthamiana

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing ...

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease Cas9, a physical barrier was microsurgically inserted between the testes of Spodoptera litura to study the impact of this microsurgery on its growth and reproduction. After inserting aluminum foil between the testes, insect molting during metamorphosis proceeded normally. Insect growth and development were not remarkably altered; however, the number of sperm bundles changed if testes fusion was stopped by the microsurgery. These findings imply that blocking testicular fusion can influence male reproduction capability. The method can be further applied to interrupt communication between organs to study the function of specific signaling pathways. Compared to conventional surgery, microsurgery only requires freezing anesthetization, which is preferable to carbon dioxide anesthetization. Microsurgery also minimizes the surgery site area and facilitates wound healing. However, the selection of materials with specific functions needs further investigation. Avoiding tissue injury is crucial when making incisions during the operation.

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Aluminum foil was microsurgically inserted between the testes of Spodoptera litura to obstruct the fusion of testis. The procedure includes freezing, fixing, disinfection, incision, placing the barrier, suturing, postoperative feeding, and inspection. This approach provides a method to interfere with tissue formation.

Ostruzione microchirurgica della fusione dei testicoli in Spodoptera litura

Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated ...