Este trabajo es apoyado por fondos de puesta en marcha de la Universidad de Louisville y CA135123 de los Institutos Nacionales de Salud (para CH).

1. Cultivo de células y transfección <ol><li> Células COS-7 se cultivaron en medio Dulbecco modificado de Eagle (DMEM) suplementado con 4,5 g / l de glucosa y 10% de suero fetal bovino (FBS). </li><li> Transfección del plásmido se lleva a cabo con Lipofectamine según las instrucciones del fabricante (Invitrogen). </li></ol> 2. Análisis de fluorescencia microscópica de la superficie celular Expresión SNARE <ol><li> El día antes de la transfección, 3 × 10 4 células COS-7 se sembraron en cubreobjetos de vidrio estériles de 12 mm (Fisher Scientific) que figuran en placas de 24 pocillos. </li><li> Para V-células en cada pocillo, 0,25 g del plásmido que codifica tTA (pTet-Off, Clontech) se cotransfectaron con 0,25 g de los plásmidos que expresan volteado VAMPS. </li><li> Para las células T en cada pocillo, 0,25 g de plásmido que codifica la TRE-LacZ (pBI-G, Clontech) se cotransfectaron con 0,25 g de cada uno de los plásmidos que expresan volteado SNAP-25 y syntaxins 1 o 4. </li><li> 24 horas después de transfection, las células se fijan con 4% de paraformaldehído en PBS + + (PBS suplementado con 0,1 g / l CaCl 2 y 0,1 g / l MgCl 2) durante 10 min, y después se bloquearon en FBS al 10% en PBS + + de 30 min. </li><li> Las células se incubaron durante 1,5 h con las 9E10 anticuerpos monoclonales anti-Myc (sobrenadante del cultivo de hibridoma puro). </li><li> Después de cuatro lavados con PBS + +, las células se incubaron durante 1 h con FITC conjugado con anticuerpos secundarios (Jackson ImmunoResearch Laboratories) a una dilución de 1:500. </li><li> Después de cuatro lavados con PBS + +, las células marcadas se montan en Prolong antifade reactivo Gold (Invitrogen). </li><li> Imágenes confocales se recogen en un láser confocal de barrido microscopio Olympus. Las imágenes se procesaron con el software Adobe Photoshop. </li></ol> 3. FACS Análisis de la expresión SNARE superficie celular <ol><li> El día antes de la transfección, 2 × 10 5 células COS-7 se sembraron en cada pocillo de placas de 6 pocillos. </li><li> 24 horas después del transfection con el volteado SNARE, pTet-Off y plásmidos pBI-G, las células se fijaron con paraformaldehído al 1% en PBS + + durante 15 min, y después se bloquearon en FBS al 10% en PBS + + de 15 min. </li><li> Las células se incubaron con los anticuerpos 9E10 monoclonales anti-Myc por 60 min. </li><li> Después de tres lavados con PBS + +, las células se marcan con FITC conjugado con anticuerpos secundarios (1:200 dilución) durante 45 min. </li><li> Después de tres lavados con PBS + +, las células se raspan de las placas con un raspador de células. </li><li> 15.000 células se analizaron utilizando un citómetro de flujo FACSCalibur (BD Biosciences). La intensidad media de fluorescencia de cada muestra se obtuvieron usando el software CellQuest Pro. </li></ol> 4. Ensayo de fusión célula enzimática <ol><li> El día antes de la transfección, 1,2 x 10 6 células COS-7 se siembran en cada placa de cultivo tisular 100-mm cultura, y 2 × 10 5 células COS-7 se sembraron en cada pocillo de placas de 6 pocillos. </li><li> Por v-células, 0,5 - 5 mg cada una de flipped plásmido VAMP se cotransfectaron con 5 g de pTet-Off en las células en cada placa de cultivo de 100-mm. Control de las células se cotransfectaron con el vector pcDNA3.1 vacío (+) y pTet-Off. Para evitar que la N-glicosilación de VAMPS 1, 4, 5, 7 y 8, tunicamicina (10 mg / ml) se incluye en medio de cultivo celular durante la transfección. </li><li> Para las células T en cada pocillo de las placas 6-y, 1 g de volteado SNAP-25 plásmido pBI-G y se cotransfectan con 0,5 ug de plásmido syntaxin1 volteado o 0,05 g de volteado syntaxin4 plásmido. </li><li> 24 horas después de la transfección, las células en V se desprenden de las placas de cultivo con la enzima libre de disociación celular Buffer (Invitrogen). Independiente células se cuentan con un hemocitómetro y se resuspendieron en HEPES tamponada con DMEM suplementado con FBS al 10%, 6,7 mg / ml tunicamicina y 0,67 mM de DTT. </li><li> 4,8 × 10 5 v-resuspendieron las células se añaden a cada pocillo que ya contenía las células-T. </li><li> Después de 6, 12 o 24 hr rata 37 ° C en 5% de CO 2 </sub&gt;, la expresión de β-galactosidasa se midió utilizando el sistema de enzimas β-galactosidasa de ensayo con tampón de lisis reportero de acuerdo con las instrucciones del fabricante (Promega). Brevemente, las células se lavan dos veces con PBS, y después se lisaron en tampón de lisis Reporter. Los lisados ​​celulares se mezclaron con un volumen igual de tampón de ensayo × 2. Como control en blanco, el tampón de lisis Reporter se mezcla con el tampón de ensayo × 2. </li><li> Después de 90 min, la reacción colorimétrica se detiene mediante la adición de carbonato de sodio 1 M. </li><li> La absorbancia a 420 nm se midió utilizando un espectrofotómetro HITACHI 100-40. </li></ol>

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No hay conflictos de interés declarado.

El ensayo de células fusión original 6 determina mediadas SNARE-eventos de fusión de células mediante microscopía de fluorescencia. Aquí se describe un ensayo que cuantifica innovador SNARE mediada por los acontecimientos de fusión celular activado por la expresión de β-galactosidasa y la medición espectrométrica. Usando este ensayo, se analizan rutinariamente 15 a 20 y v-t-SNARE combinaciones en un solo experimento. Usando citometría de flujo para medir la expresión SNARE en la superficie celular, que valorar los niveles de expresión de VAMPS y comparar sus capacidades de fusión de membranas (Figuras 2 y 4). Además, usando el ensayo de fusión celular enzimática, se determinó la dependencia de la actividad de fusión de células de la densidad de la superficie celular de VAMP1 proteína, y no observaron ninguna cooperatividad de VAMP1 proteína en la reacción de fusión de 8 células, lo que sugiere que la acción concertada de complejos SNARE múltiples no se requiere para fusionar las membranas celulares. Hay putativo N-glicosilación Motifs (Asn-X-Ser/Thr) en las proteínas SNARE. Debido a la N-glicosilación inhibe las actividades de fusión de volteado proteínas SNARE 6, dos enfoques se han utilizado para prevenir la glicosilación de las proteínas SNARE volteado. En primer lugar, el sitio de glicosilación de las mutaciones se introducen en volteado SNARE construye 6. En segundo lugar, como se describe en el protocolo actual, la tunicamicina inhibidor de N-glicosilación (6,7 g / ml) se incluyó en la reacción de la fusión celular. Además, 0,67 mM DTT, se añade en la reacción de fusión celular para evitar la formación de enlaces disulfuro entre volteado proteínas SNARE, que pueda inhibir las actividades de SNARE de fusión. Nuestros experimentos muestran que la adición de 6,7 g / ml tunicamicina y 0,67 mM de DTT en medio de cultivo celular no interfiere con la proliferación y supervivencia de células COS-7. En forma rutinaria terminar la reacción de fusión celular a las 24 h después de la combinación y v-t-células. Sin embargo, significativa SNARE mediada por la fusión de células se detecta 6 hr después de combinar v-aND T-células 8. Debido a que la reacción de fusión celular enzimática depende de la expresión de β-galactosidasa después de la fusión celular, el curso temporal de este sistema de lectura se retrasa respecto al transcurso del tiempo de la fusión de membranas mediada por volteado proteínas SNARE. El ensayo de fusión celular enzimática ofrece un ensayo de reconstitución cuantitativa del examen de las actividades de fusión de membranas de potencial V-y t-SNARE interacciones en células de mamífero. Por coexpresan proteínas reguladoras (por ejemplo, Munc18) y SNAREs en la superficie celular o mediante la inclusión de proteínas recombinantes de regulación en la reacción de fusión celular, este ensayo puede ser utilizada para aclarar los mecanismos de regulación de la fusión de membranas mediada por SNARE. Además, este ensayo se deben tener aplicaciones en la selección de alto rendimiento para inhibidores o activadores de la fusión de membranas mediada por SNARE.

<table border="1"><tbody><tr><td> Nombre de los reactivos y equipos </td><td> Empresa </td><td> El número de catálogo </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12800-017 </td></tr><tr><td> COS-7 células </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 libre de células solución de disociación </td><td> Invitrogen </td><td> 13151-014 </td></tr><tr><td> De conejo anti-represor Tet anticuerpo policlonal </td><td> Millipore </td><td> AB3541 </td></tr><tr><td> FITC-conjugado con burro anti-ratón IgG (H + L) </td><td> JacKson ImmunoResearch </td><td> 715-095-150 </td></tr><tr><td> El escaneo láser confocal microscopio </td><td> Olimpo </td><td> FV1000 </td></tr><tr><td> FACSCalibur citómetro de flujo </td><td> BD Biosciences </td><td></td></tr><tr><td> β-galactosidasa sistema de ensayo con tampón de lisis reportero </td><td> Promega </td><td> E2000 </td></tr><tr><td> Modelo 100-40 espectrofotómetro UV-VIS </td><td> Hitachi </td><td> C740843 </td></tr></tbody></table>

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

Las interacciones de las SNARE (s oluble N-etilmaleimida factor sensible a una proteína ttachment ceptor re) proteínas en vesículas (v-SNARE) y en membranas diana (t-SNARE) catalizan la fusión de vesículas intracelulares 1-4. Ensayos de reconstitución son esenciales para disecar el mecanismo y la regulación de la fusión de membranas mediada por 5-SNARE. En un ensayo de fusión celular 6,7, las proteínas SNARE se expresan ectópicamente en la superficie celular. Estos &quot;volteado&quot; SNARE unidad proteínas de fusión célula-célula, lo que demuestra que trampas son suficientes para fusionar las membranas celulares. Debido a que el ensayo de fusión celular se basa en el análisis microscópico, que es menos eficiente cuando se usa para analizar múltiples interacciones y v-t-SNARE cuantitativamente. Aquí se describe un nuevo ensayo que cuantifica 8 SNARE mediada por los acontecimientos de fusión celular activado por la expresión de β-galactosidasa. Doscomponentes del sistema de genes Tet-Off expresión 9 se utiliza como un sistema de lectura: el transactivador controlado por tetraciclina (tTA) y un plásmido informador que codifica el gen LacZ bajo el control del elemento de respuesta de tetraciclina (TRE-LacZ). Nos transfectar tTA en células COS-7 que expresan volteado v-SNARE proteínas en la superficie celular (V-células) y transfección de TRE-LacZ en células COS-7 que expresan volteado t-SNARE proteínas en la superficie celular (células T) . SNARE dependiente de la fusión de la V-y las células T resulta en la unión de tTA a TRE, la activación transcripcional de LacZ y la expresión de β-galactosidasa. La actividad de β-galactosidasa se cuantificó usando un método colorimétrico por absorbancia a 420 nm. Las proteínas asociadas a la membrana de vesículas (vampiros) son v-SNARE que residen en diferentes compartimentos de Golgi post-vesiculares 10-15. Mediante la expresión de VAMPS 1, 3, 4, 5, 7 y 8 en el mismo nivel, se compara su fusión de membranasactividades utilizando el ensayo de células de fusión enzimática. Sobre la base de la medición espectrométrica, este ensayo ofrece un enfoque cuantitativo para el análisis de SNARE mediada por fusión de membranas y para estudios de alto rendimiento.

Para desarrollar un ensayo de fusión celular cuantitativos, se aprovecha de la fuerte activación transcripcional por la unión de tTA a TRE. En ausencia de tTA, la transcripción del gen LacZ en TRE-LacZ está en silencio. Cuando tTA está presente, se une a la TRE y activa la transcripción de lacZ. Figura 1 muestra un diagrama de flujo de la fusión de células de ensayo enzimático. tTA se transfecta en v-células que expresan v-SNARE proteínas en la superficie celular, y TRE-LacZ se transfecta en células T que expresan t-SNARE proteínas en la superficie celular. Fusión de la V-y las células T resulta en la unión de tTA a TRE, la activación de la transcripción de lacZ, y la expresión de β-galactosidasa. La Figura 2A ilustra la estructura de dominio de las construcciones de SNARE volteado. La secuencia señal preprolactin se fusiona al N-terminales de las trampas. Estas trampas de ingeniería se llaman &quot;volteado&quot;SNAREs porque la orientación de sus motivos SNARE en contra de las membranas celulares se da la vuelta. Cuando células COS-7 se transfectaron con plásmidos volteado SNARE, volteado proteínas SNARE se expresan en la superficie celular (Figura 2B). La citometría de flujo se utiliza para medir los niveles de expresión de las proteínas SNARE en la superficie celular (Figuras 2C y D). Con el fin de comparar sus capacidades de fusión de membrana, VAMPS necesitan ser expresados ​​en el mismo nivel. Por lo tanto, se valoraron y se optimizó la concentración de cada volteado SNARE plásmido utilizado en la transfección. Flipped SNARE plásmidos se transfectaron en las siguientes concentraciones (por cada 10 cm 2 área de crecimiento, es decir, por pocillo en placas de 6 pocillos): VAMP1, 0,2 g; VAMP3, 0,5 g; VAMP4, 0,5 g; VAMP5, 0,05 g; VAMP7, 1,0 g; VAMP8, 0,1 g; syntaxin1, 0,5 g; syntaxin4, 0,05 g. TTA, TRE-LacZ y se volcó SNAP-25 se cotransfectaron a 1 g por 10 cm zona de crecimiento 2. Bajo éxitoh Condiciones, VAMPS 1, 3, 4, 5, 7 y 8 se expresan en el mismo nivel, mientras que syntaxins 1 y 4 se expresan en el mismo nivel en la superficie celular (Figura 2D). Como se muestra por análisis de contraste de la imagen confocal y fase (2E Figura), 80% de las células COS-7 se transfectaron con plásmidos volteado SNARE. Análisis de imágenes de doble etiquetado (Figura 2F) indica que el 70% de las células transfectadas v-expresar tanto tTA y volteado proteínas VAMP. Debido a que el gen LacZ en TRE-LacZ no se expresa antes de la fusión celular, que no son capaces de determinar el porcentaje de transfectado las células T que expresan tanto TRE-LacZ y se volcó t-SNARE proteínas. Las trampas neuronales (v-SNARE VAMP2 y syntaxin1 t-SNARE y SNAP-25) que median la exocitosis sináptica se utilizan para probar la viabilidad de la prueba. En efecto, cuando las células que expresan v-VAMP2 y las células T que expresan syntaxin1/SNAP-25 se combinan, robusto β-Galactosidase expresión se detecta (Figura 3). Sin embargo, cuando sea VAMP2 o SNAP 25-no se expresa, sólo basal β-galactosidasa se detecta actividad, lo que indica que la fusión celular y la expresión de β-galactosidasa se basan en interacciones de v-y t-SNARE. Estos resultados indican que el ensayo de fusión celular enzimática identifica emparejamientos fusogénicos entre V y t-SNARE eficiente. Mediante la expresión de las proteínas VAMP en el mismo nivel en la superficie celular (Figura 2D), se comparan sus actividades de fusión de membranas. Con el t-SNARE syntaxin1/SNAP-25, VAMPS 1, 3, y 8 tienen comparable y las actividades más altas de fusión, mientras que VAMPS 4 y 7 tienen 50% y 30% más bajas actividades de fusión, respectivamente (Figura 4). En contraste, cuando VAMP5 se combina con el t-SNARE, sólo basal β-galactosidasa se ​​detecta actividad (Figura 4), ​​lo que sugiere que VAMP5 no conduce fusión de la membrana con el Syntaxin1/SNAP-25. <img alt="Figura 1" src="/files/ftp_upload/4378/4378fig1.jpg" /> Figura 1. Ensayo enzimático de la fusión celular. tTA se cotransfectaron con volteado v-SNARE plásmidos, y TRE-LacZ se cotransfectó con volteado t-SNARE plásmidos. La fusión de tipo V y las células T conduce a la unión de tTA a TRE y la expresión de β-galactosidasa, que se mide mediante un método colorimétrico. <img alt="Figura 2" src="/files/ftp_upload/4378/4378fig2.jpg" /> Figura 2. Expresión de volteado SNAREs en la superficie celular. (A) la estructura de dominio de volteado trampas. La secuencia señal preprolactin (SS) se fusiona a los extremos N de las trampas, y una etiqueta Myc se inserta entre la secuencia señal y los motivos SNARE (espiral de la bobina). (B) células COS-7 se cotransfectan con tTA y el vector vacío pcDNA3.1 (+) o volteado VAMP5 plásmido. 24h después de la transfección, las células unpermeabilized se tiñeron con un anticuerpo monoclonal anti-Myc. Representante de un único segmento confocal de imágenes se muestran. (C y D) 24 hr después de la cotransfección con tTA y pcDNA3.1 (+) o volteado Vamps (V-células), o 24 horas después de la cotransfección con TRE-LacZ, volteado SNAP-25 y syntaxins 1 o 4 (T-células ), las células unpermeabilized se tiñeron con el anticuerpo anti-Myc y se analizaron por citometría de flujo. (C) representativos perfiles FACS de las células transfectadas con pcDNA3.1 (+) o volteado VAMP1 plásmido. La intensidad media de fluorescencia de cada muestra se determina usando el software CellQuest Pro. (D) Para expresar VAMPS en el mismo nivel y expresa syntaxins 1 y 4 en el mismo nivel en la superficie celular, volteado SNARE plásmidos se transfectaron en concentraciones valoradas. Se muestran las intensidades medias de fluorescencia de la tinción de la superficie celular de las proteínas SNARE. Las barras de error representan la desviación estándar de 4 experimentos independientes. (E y F) 24 hr después de cotransfe<html> cción con tTA y volteado VAMP5 plásmido, las células se permeablized con 0,2% Triton X-100 en PBS + + y (E) se tiñeron con el anticuerpo anti-Myc, o (F) manchado dual con el anticuerpo monoclonal anti-Myc y un anticuerpo policlonal el represor TET (para detectar tTA). (E) que se muestran son representativas confocal y las imágenes de contraste de fase. El número de células transfectadas que están marcados por el anticuerpo anti-Myc (VAMP5) y el número total de células en el canal de contraste de fase se cuentan (n = 60 células), y la eficiencia de la transfección se calcula (80%). (F) El número de células transfectadas que expresan tanto VAMP5 y tTA, y el número total de células transfectadas que expresan VAMP5, tTA o ambos se cuentan (n = 75 células transfectadas). El porcentaje de las células transfectadas que expresan tanto VAMP5 tTA y se calcula a continuación (70%). Las barras de escala, a 20 m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/4378/4378fig2large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . &quot;Fo: keep-together.within-page =&quot; always &quot;&gt; <img alt="Figura 3" src="/files/ftp_upload/4378/4378fig3.jpg" /> Figura 3. Fusión celular depende de las interacciones de v-y t-SNARE. v-células que expresan tTA y VAMP2 se incuban con las células T que expresan TRE-LacZ, syntaxin1 y SNAP 25-. Después de 24 h, las células se lisaron y la actividad de β-galactosidasa en los lisados ​​de células se determinó utilizando un método colorimétrico por absorbancia a 420 nm. Línea de base sólo b-galactosidasa se detecta actividad cuando sea volteado VAMP2 o SNAP-25 plásmido se omite de la transfección. <img alt="Figura 4" src="/files/ftp_upload/4378/4378fig4.jpg" /> Figura 4. Comparación de las actividades de fusión de vampiros. Bajo las condiciones de transfección optimizadas, VAMPS 1, 3, 4, 5, 7 y 8 se expresan en el mismo nivel en la superficie celular de las células v. 24 horas después de la combinación de los v-células con las células T que exsyntaxin1/SNAP-25 prensa, la fusión celular se cuantificó usando el ensayo de fusión celular enzimática. Las barras de error representan la desviación estándar de 4 experimentos independientes. ** P &lt;0,01; *** P &lt;0,001 vs VAMP1.

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

Hemos desarrollado un ensayo de fusión celular que cuantifica SNARE mediada por los acontecimientos de fusión de membrana por la expresión activa de β-galactosidasa.

Análisis de la fusión de la membrana SNARE mediada usando un ensayo de fusión celular enzimática

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

Nanyang Technological University

Research

JoVE Journal

Biology

13.9K vistas.  University of Louisville School of Medicine. Hemos desarrollado un ensayo de fusión celular que cuantifica SNARE mediada por los acontecimientos de fusión de membrana por la expresión activa de β-galactosidasa.

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

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

El estudio de biogénesis de la membrana con un ensayo de luciferasa basado en genes

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Investigación

Biología

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.

Método para la medición de la cinética de fusión viral a nivel de partícula

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

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.

La discectomía cervical anterior con fusión en el modelo ovino

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.

Marcaje fluorescente de COS-7 Expresando etiqueta SNAP-proteínas de fusión de imágenes de células vivas

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 más rápida, de alta resolución, APMt-GFP-ensayo basado en la fusión mitocondrial Adquisición de datos cinéticos de varias celdas en paralelo mediante microscopía confocal

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.

Expresión de proteínas fluorescentes de fusión en murinas médula ósea células dendríticas y macrófagos

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.

Ensayo de absorción de colesterol LDL utilizando celular directo imágenes análisis con células control de salud

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.

Efecto de proteínas fluorescentes de fusión socios en ensayos de toxicidad de poliglutaminas de levadura

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.

Explotando en la proyección de imagen a los núcleos de la pista durante la fusión y diferenciación de mioblastos

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.

Producción de proteínas de fusión IgG expresada transitoriamente en Nicotiana benthamiana

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