Gracias al Dr. Praveen Kumar en el laboratorio de Wittman en la UCSF para la película de la actina en células HaCaT.

1. Preparación de la actina La fuente de la actina utilizada en este ensayo no era muscular plaquetas humanas (β-actina) obtenidos a partir del citoesqueleto Inc. alícuotas de archivo de 10 mg / ml se mantienen en el congelador -70 º C. Para el ensayo, la actina se diluye a 0,4 mg / ml en un tampón que contenía 5 mM Tris pH 8, 0,2 mM CaCl 2, 0,2 mM ATP y 0,5 mM de TDT, se centrifuga a 20.000 g durante 10 minutos a 4 º C. El sobrenadante, que es la actina monomérica está listo para ser polimerizado. Polimerización de la actina es inducida por la adición de KCl 50 mM, 1 mM ATP y 2 mM de MgCl 2. La polimerización se produce a temperatura ambiente durante 1 hora. 2. Preparación de la proteína En este ensayo, se utiliza el C-terminal de Tallin, que se ha purificado como una proteína GST-etiquetados. La proteína que se utiliza en el ensayo se somete a una centrifugación de alta velocidad para pre-clara de los agregados antes de la incubación con F-actina. La proteína se hace girar a 100.000 g en una ultracentrífuga Beckman durante 20 minutos a 22 º C. 3. Actina-proteína de incubación La cantidad de actina y las proteínas en un ensayo puede variar. En este ejemplo estamos utilizando actina en exceso. Normalmente, una relación molar se calcula, por ejemplo. una relación de 4:1 molar de actina a la proteína. En el siguiente ensayo, el volumen final de reacción es de 150 l. Búfer en el que se incuban las proteínas y F-actina dependerá de la proteína a ser probado y en las condiciones requeridas. En este ejemplo, se utiliza un tampón que contiene 10 mM Tris pH 7,0, 1 mM ATP, 0,2 mM DTT, 1 mM EGTA, 0,1 mM CaCl 2 y 2 mM de MgCl 2. La proteína y el F-actina se incuban en el buffer durante 1 hora a temperatura ambiente. 4. La sedimentación de la F-actina Después de la incubación, las muestras se centrifugaron a 100.000 g a 22 º C. Utilizamos una ultracentrífuga Beckman en el video. El rotor se retira cuidadosamente para no molestar a los pellets. 5. El análisis de proteínas co-sedimentación con F-actina Los sobrenadantes se retira cuidadosamente con la pipeta en un tubo eppendorf y 5 X Laemmli SDS-PAGE muestra de amortiguación, se añade. Un volumen adecuado de 1 x tampón de muestra Laemmli SDS-PAGE se añade al resto de pellets, una pipeta de arriba a abajo, y trasladado a nuevos tubos eppendorf. Las cantidades relativas de proteína en los sedimentos y sobrenadantes son analizados después de su separación por SDS-PAGE y tinción con azul de Coomassie del gel o Western Blot. Con el fin de determinar la especificidad de la interacción con la proteína actina, la actina dependiente de la concentración de co-sedimentación se puede realizar. Para este tipo de experimento, una cantidad fija de la proteína y una serie de cantidades cada vez mayores de la actina se incuban, y el análisis se lleva a cabo como se describió anteriormente.

<ol>
	<li>Senetar, M. A., Foster, S. J., McCann, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrasteric+inhibition+mediates+the+interaction+of+the+I/LWEQ+module+proteins+Talin1,+Talin2,+Hip1,+and+Hip12+with+actin.">Intrasteric inhibition mediates the interaction of the I/LWEQ module proteins Talin1, Talin2, Hip1, and Hip12 with actin.</a> Biochemistry. 14, 15418-15428 (2004).</li><li>Goldmann, W. H., Hess, D., Isenberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+intact+talin+and+talin+tail+fragment+on+actin+filament+dynamics+and+structure+depends+on+pH+and+ionic+strength.">The effect of intact talin and talin tail fragment on actin filament dynamics and structure depends on pH and ionic strength.</a> Eur J Biochem. 260, 439-445 (1999).</li><li>Lee, H., Bellin, R. M., Walker, D. L., Patel, B., Powers, P., Liu, H., Garcia-Alvarez, B., de Pereda, J., Liddington, R. C., Volkmann, N., Hanein, D., Critchley, D. R., Robson, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+Actin-binding+Site+within+the+Talin+FERM+Domain.">Characterization of an Actin-binding Site within the Talin FERM Domain.</a> J Mol Biol. 22, 771-784 (2004).</li></ol>

Actina co-sedimentación es un simple ensayo in vitro para analizar las proteínas de unión a specfic F-actina. En este vídeo, que muestran un ejemplo de cómo un sencillo actina co-sedimentación puede llevarse a cabo. Se muestra cómo preparar la F-actina, preparar la proteína a ser probado y el procedimiento de la actina co-sedimentación. Una serie de puntos deben ser considerados en el ejercicio de actina sedimentación co-ensayos. Por ejemplo, los componentes del tampón de incubación puede variar en función de la proteína a ser probado y el pH, la temperatura y la concentración de sal puede afectar la unión de F-actina. Hemos demostrado un enlace simple de F-actina, sin embargo este tipo de ensayo puede ser elaborado y utilizado para determinar la especificidad de unión de un dominio de la proteína y proteína para la F-actina.

actin co-sedimentation assay; for the analysis of protein binding to f-actin

El citoesqueleto de actina en la célula es una red de filamentos de actina que permite el movimiento de las células y los procesos celulares, y que genera tensión y ayuda a mantener la forma celular. Aunque el citoesqueleto de actina es una estructura rígida, es una estructura dinámica que está en constante remodelación. Una serie de proteínas se puede unir al citoesqueleto de actina. La unión de una proteína en particular a la F-actina se desea a menudo para apoyar la célula observaciones biológicas o para comprender mejor los procesos dinámicos debido a la remodelación del citoesqueleto de actina. La actina co-sedimentación ensayo es un ensayo in vitro utilizan habitualmente para analizar la unión de proteínas específicas o dominios de la proteína con la F-actina. Los principios básicos de la prueba implica un período de incubación de la proteína de interés (de larga duración o el dominio de), con F-actina, paso ultracentrifugación para precipitar la F-actina y el análisis de la proteína co-sedimentación con F-actina. Actina sedimentación co-ensayos pueden ser diseñados para medir la afinidad de unión de actina y en los ensayos de la competencia.

Watch this Scientific Journal Video about Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin at JoVE.com

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

Proteínas se unen a la actina filamentosa (F-actina) a través de módulos de unión de actina distintas. En este video se demuestra el procedimiento de la actina co-sedimentación, que es un ensayo in vitro utilizan habitualmente para analizar las proteínas o dominios específicos que se unen a la F-actina.

Actina Co-sedimentación de ensayo, pues el análisis de proteína de unión a la F-actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

actin-co-sedimentation-assay-for-analysis-protein-binding-to-f

Nanyang Technological University

Research

JoVE Journal

Biology

19.3K vistas.  University of California, San Francisco - UCSF. Proteínas se unen a la actina filamentosa (F-actina) a través de módulos de unión de actina distintas. En este video se demuestra el procedimiento de la actina co-sedimentación, que es un ensayo in vitro utilizan habitualmente para analizar las proteínas o dominios específicos que se unen a la F-actina.

Video: Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

Medición de la unión de proteínas a actina F por co-sedimentación

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Investigación

Bioquímica

In Vitro Polymerization of F-actin on Early Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5. Cytosolic fractions are prepared from separate batches of cells. Both endosomal and cytosolic fractions can be stored frozen in liquid nitrogen, if needed. In the assay, the endosomal and cytosolic fractions are mixed, and the mixture is incubated at 37 &#176;C under appropriate conditions (e.g., ionic strength, reducing environment). At the desired time, the reaction mixture is fixed, and the F-actin is revealed with phalloidin. Actin nucleation and polymerization are then analyzed by fluorescence microscopy. Here, we report that this assay can be used to investigate the role of factors that are involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of F-actin filaments.

In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations.

In Vitro Polimerización de la F-actina en endosomas tempranos

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

Desde un 2DE-Gel Spot para Función de Proteínas: Lección aprendida de HS1 en la leucemia linfocítica crónica

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Medicina

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

Uso de microfluídica y microscopía de fluorescencia para estudiar la dinámica de ensamblaje de filamentos y haces de actina única

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.&#160;As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

Ensayo de sedimentación basado en concanavalina A para medir la unión al sustrato de las fosfatasas de glucano

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...

Reconstitution of Actin-Based Motility with Commercially Available Proteins

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a dynamic network at the surface of the cell, organelle, or bacterium. The biochemical and mechanical basis of force production during this process can be studied by reproducing actin-based movement in an acellular manner on inert surfaces such as beads that are functionalized and incubated with a controlled set of components. Under the appropriate conditions, an elastic actin network assembles at the bead surface and breaks open due to the stress generated by network growth, forming an "actin comet" that propels the bead forward. However, such experiments require the purification of a host of different actin-binding proteins, often putting them beyond the reach of non-specialists. This article details a protocol for reproducibly obtaining actin comets and motility of beads using commercially available reagents. Bead coating, bead size, and motility mixture can be altered to observe the effect on bead speed, trajectories, and other parameters. This assay can be used for testing the biochemical activities of different actin-binding proteins, and for performing quantitative physical measurements that shed light on active matter properties of actin networks. This will be a useful tool for the community, enabling the study of in vitro actin-based motility without expert knowledge in actin-binding protein purification.

This protocol describes how to produce actin comets on the surfaces of beads using commercially available protein ingredients. Such systems mimic the protrusive structures found in cells, and can be used to examine physiological mechanisms of force production in a simplified way.

Reconstitución de la motilidad basada en actina con proteínas disponibles comercialmente

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a ...

Bioingeniería

FtsZ Polymerization Assays: Simple Protocols and Considerations

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization.

Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.

FtsZ Polimerización Ensayos: Protocolos simples y Consideraciones

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long ...

Biología

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

Un Ensayo Basado En Espectroscopia De Fluorescencia Eficiente En El Tiempo Para Evaluar El Estado De Polimerización De Actina En Roedores Y Tejidos Cerebrales Humanos

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

Neurociencias

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

Análisis de la dinámica de la actina, el acoplamiento del embrague y la fuerza de tracción para el avance del cono de crecimiento

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

Reconstitución de corticidades mínimas de actina atadas a la membrana en bicapas lipídicas soportadas

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...