C.G. is supported by grants from the Agence National de la Recherche (ANR-13-JSV1-0008), from the European Union Seventh Framework Programme (Marie Curie Career Integration n&#730;8304162) and from European Research Council (ERC) under European Union&#39;s Horizon 2020 research and innovation program (ERC Starting Grant n&#730;639300).

1. Ligand Conjugation to Magnetic Beads
Note: Ligand conjugation is performed using superparamagnetic tosyl-activated beads with a 2.8 &#956;m diameter (stock solution concentration 108 beads/mL, 30 mg beads/mL). The following protocol is based on samples of approximately 2 x 105 cells, which correspond to MRC-5 cells grown to 80% confluency in a 60 mm tissue culture plate. Adjust the volume of beads and reagents accordingly if using plates of different sizes or cells at different confluences. Use an amount of superparamagnetic beads in order to have 2 beads per cell. Therefore, 4 x 105 beads are needed for a 60 mm plate.
<ol>
	<li>Thoroughly resuspend the beads in the original vial by vortexing at least 30 s. Aliquot 40 &#181;L of the resuspended superparamagnetic beads (corresponding to 4 x 106 beads) to 1.5 mL microcentrifuge tube.</li>
	<li>Place the tube on a magnetic separation stand in order to separate the magnetic beads from the solution. Discard the supernatant, remove tube from the magnetic separation stand and resuspend the beads in 1 mL 0.1 M Na-phosphate pH 7.4.
	<ol>
		<li>Repeat wash step twice with 1 mL 0.1 M Na-phosphate pH 7.4.</li>
	</ol>
	</li>
	<li>After the final wash, resuspend the beads in 1 mL 0.1 M Na-Phosphate pH 7.4.</li>
	<li>Combine 100 &#956;g Bovine Fibronectin (FN) or any other cell adhesion receptor ligand to the 1 mL 0.1 M Na-phosphate pH 7.4 containing beads and mix by pipetting.</li>
	<li>Repeat previous steps by replacing FN or any other specific ligand with BSA, poly-D-lysine or apo-transferrin for negative controls.</li>
	<li>Optionally, for gel analysis, take a 10 &#956;L aliquot of ligand solution for analysis of crosslinking efficiency and mix with an appropriate volume of concentrated Laemmli Sample Buffer.</li>
	<li>Incubate beads with the ligand containing solution for 12-24 h at 37 &#176;C on a rotor.</li>
	<li>If bead aggregates appear after the reaction has occurred, sonicate (continuous 39 W power) for no more than 10-20 s.</li>
	<li>Isolate the beads using magnetic separation stand, aspirate the remaining solution and add 1 mL of PBS/0.2% BSA pH 7.6 solution. Incubate for 1 h on rotor at 37 &#176;C.</li>
	<li>Wash beads using a magnet twice with 1 mL PBS/0.2% BSA pH 7.6. Resuspend beads in 1 mL PBS/0.2% BSA pH 7.6.</li>
	<li>Sonicate beads if aggregates appear for no more than 20-30 s.</li>
	<li>Optionally, remove a 10 &#956;L aliquot to analyze coupling efficiency by western blot (mix with an appropriate volume of concentrated Laemmli Sample Buffer).</li>
	<li>Proceed to cell assays or store beads at 4 &#176;C for up to 1 month.</li>
	<li>Optionally, run a SDS-PAGE gel and stain with Coomassie blue to analyze FN crosslinking to the magnetic beads.</li>
</ol>
2. Application of Tensional Forces on the Ligand-coated Beads Bound to Adhesion Receptors on the Dorsal Surface of Cells
<ol>
	<li>Culture adherent cells on 60 mm tissue culture dish in appropriate growth medium (usually DMEM 4.5 g/L D-glucose supplemented with FCS 10%) until reaching 80% confluency.</li>
	<li>Prepare non-denaturing non-ionic lysis buffer (20 mM Tris HCl pH 7.6, 150 mM NaCl, 2 mM MgCl2, 0.1% NP-40) and chill microcentrifuge tubes.</li>
	<li>Pellet the ligand-coated beads from section 1.10 and resuspend in 1 mL PBS/0.2% BSA pH 7.6. Add 100 &#956;L of the ligand-coated beads solution to 5 mL of warm culture&#160;medium and vortex.</li>
	<li>Aspirate medium in 60 mm culture dish and add 5 mL warm growth medium supplemented with beads.&#160;</li>
	<li>Incubate for 20 min under cell culture conditions (37 &#176;C and 5% CO2) to allow the beads to sediment and adhere to the cells. It is crucial to not exceed 20 min since beads may be internalized by phagocytosis.</li>
	<li>Optionally, observe the beads under a light microscope using a 10X or 20X objective and check for bead adhesion by slightly shaking the dish to discriminate between attached and floating beads.</li>
	<li>While keeping the culture dish in the incubator, swap the normal dish lid for a lid with a round 38 mm neodymium magnet attached on the upper face. The magnet is held in place by two smaller 13 mm neodymium magnets positioned on the lower face of the lid. &#160;Since the magnets are very powerful, manipulate them carefully.</li>
	<li>Incubate cells subjected to tension for the desired time points at cell culture conditions (37 &#176;C and 5% CO2).&#160;</li>
	<li>After treatment, place dish on ice and carefully aspirate the entire medium from the dish.</li>
	<li>Add 300 &#956;L of cell lysis buffer, and incubate for 10 min on ice. Collect the lysate using a cell scraper and transfer to a pre-chilled 1.5 mL microcentrifuge tube.</li>
	<li>Pellet the magnetic beads using the magnetic separation stand and transfer the total cell lysate to a new pre-chilled 1.5 mL microcentrifuge tube. Store this fraction at -20 &#176;C for further analysis.</li>
	<li>Wash the beads 3 times with 1 mL ice-cold lysis buffer. Add 50 &#956;L Laemmli Sample buffer to the bead pellet, mix by pipetting and boil at 95 &#176;C for 5 min using a dry block heater. This fraction contains the isolated adhesion complexes. Proceed to biochemical analysis or store at -20 &#176;C.</li>
	<li>From the total cell lysate obtained after beads separation (about 300 &#956;L), remove a 50 &#956;L aliquot for western blot analysis. 
	NOTE: The 250 &#956;L left can be used for further biochemical studies such as protein-protein interaction studies (immunoprecipitation, GST pull-down assays) or GTPase activity experiments (changing the lysis buffer may be necessary depending on the GTPase of interest).</li>
</ol>

<ol>
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The authors declare no competing financial interests.

The method described here constitutes a straightforward approach to apply tension to cell surface adhesion receptors and allow their subsequent purification. However, some steps are critical to perform efficient adhesion purification and potential optimization can be done depending on the targeted adhesion receptors. We present potential issues the user may encounter below.
We used 2.8 &#956;m diameter magnetic beads but larger beads can be used such as 4.5 &#956;m diameter. However, bead diam...

In metazoa, mechanical tension directs tissue development and homeostasis through the regulation of a myriad of cellular processes such as proliferation, differentiation and survival 1,2. Mechanical tension can arise from the extracellular matrix or can be generated by adherent cells, which sample their extracellular environment through the actomyosin contractile machinery that pulls onto extracellular matrix and probes its rigidity through tension-sensitive molecules. In response to tension, mechanosensitive adhesion proteins undergo conformational changes that trigger complex signaling cascades. In turn, these signaling pathways orchestrate a mechanoresponse encompassing proliferation, differentiation and survival that adjusts the cellular behavior to the extracellular environment. Such processes can be settled in a short-term time period (seconds to minutes) to quickly feed back onto the loop of mechanotransduction by modifying the mechanosensitive structures. For instance, integrin-based adhesions reinforce in response to tension through Rho GTPase-mediated cytoskeletal remodeling 3,4,5. In parallel, other signaling pathways are activated over hours and days to control genetic programs that eventually impact cell fate 6. Whereas, many studies have highlighted the effect of matrix stiffness on cell determinism and disease development 1,2 , the precise molecular mechanisms of adhesion-mediated mechanotransduction still remain elusive.
Various approaches have been developed to study the effects of cell-generated forces or external forces on cell behavior, including flow systems, fluorescence resonance energy transfer (FRET)-tension sensors 7,8, compliant substrates 9, magnetic tweezers, optical tweezers 10 and atomic force microscopy (AFM) 11. Here we present a protocol using superparamagnetic beads to characterize mechanotransduction pathways in response to tensional forces applied to specific adhesion receptors. Superparamagnetic beads are particles that reversibly magnetize when placed in a magnetic field. Once coated with a ligand for a specific receptor, these beads provide a powerful tool to study the effects of extracellular force application. This method has been validated by several studies 3,5,12-17 and present the advantage to largely facilitate biochemical analysis on adherent cells. Using similar collagen-coated magnetic beads followed by biochemical analysis, early work reported an increase in protein tyrosine phosphorylation and RhoA activation in response to tension 5,18,19. The method described below has also been used with fibronectin (FN)-coated beads to characterize the signaling pathways downstream from tension applied to integrins 3. In this study, Guilluy et al. showed that tension activates RhoA through the recruitment of the two guanine nucleotide exchange factors (GEFs), LARG and GEF-H1, to integrin adhesion complexes. Since that, other studies have shown that GEF-H1 is recruited to adhesion complexes in response to cell-generated tension using different methods 20,21, demonstrating the robustness of the methodology described here. As a result, activated RhoA was shown to promote adhesion reinforcement, through cytoskeletal remodeling. This system was also used to explore tension applied to cell/cell adhesion receptors. Application of forces onto magnetic beads coated with the extracellular domain of E-cadherin induced an increase in vinculin recruitment similarly to integrin associated adhesion complexes 12. Collins and colleagues observed that application of tension to PECAM-1 promotes integrin and RhoA activation 13. Another experimental approach using magnetic beads is the study of tension applied to isolated nuclei. Using beads coated with antibodies against the nuclear envelope protein nesprin-1, nuclear envelope complexes were purified to show that they are dynamically regulated in response to mechanical tension 22. These results support the powerfulness of this method in the study of mechanotransduction pathways. Moreover, while flow or traction force systems stimulate general cellular processes, magnetic beads specifically target a cell adhesion receptor by using either receptor ligands 3 or monoclonal antibodies against cell surface receptor 13,15.
Another advantage of this method is the isolation of adhesion complexes through a straightforward ligand affinity purification procedure. It is well known that addition of ligand-coated beads to cells binds adhesion receptors and induces the recruitment of several adhesion proteins23. Further application of forces to ligand-coated magnetic beads turns these adhesion complexes into macromolecular platforms that mediates various tension-dependent signaling pathways4,24. Cell lysis followed by bead concentration using a magnet permits the isolation of the adhesion platforms. Other methods used to purify adhesion complexes have already been used in adherent cells. They combine chemical crosslinking to conserve protein-protein interactions and a cell lysis step by detergent and shear flow or sonication 20,21,25,26,27,28. The final step is the collection of the resulting ventral plasma membranes containing the adhesion complexes. Unlike these methods, magnetic beads allow a greater purification level of cell adhesion complexes by selectively targeting a specific family of adhesion receptors. Magnetic beads have already been used to purify adhesion complexes in non-adherent cells attached to ligand-coated microbeads29,30. The method described below mimics biological situations where force is applied for a short sustained period (seconds to minutes). Therefore, it provides a powerful tool for investigating both the molecular composition of purified adhesion complexes and the downstream mechanosensitive-signaling pathways.
Here we present a detailed experimental protocol for using magnetic beads to apply tensional forces to adhesion surface proteins. A permanent neodymium magnet is placed on top of the culture dish surface. The pole face of the magnet is placed at a height of 6 mm so that the force on a single 2.8 &#956;m magnetic bead is constant (about 30-40 pN)31. The duration of tension stimulation is determined by the operator depending on the molecule of interest and its time-scale of activation. Cells are finally lysed, adhesion complexes are purified by beads separation using a magnet and biochemical analyses are processed. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of tension through magnet followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesions induces adhesion remodeling and alters protein tyrosine phosphorylation.

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			<td style="width:103px;">DX88-N52</td>
			<td style="width:375px;">grade N52 dimension: 1 1/2&#34; dia. x 1/2&#34; thick</td>
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			<td height="42" style="height:42px;width:231px;">Neodymium magnets (on the lower face of 60 mm dish)</td>
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			<td style="width:103px;">D84PC-BLK</td>
			<td>grade N42 dimension: 1/2&#34; dia. x 1/4&#34; thick Black Plastic Coated&#160;</td>
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			<td height="21" style="height:21px;width:231px;">Dynabeads M280 Tosylactivated</td>
			<td style="width:115px;">Thermofisher</td>
			<td style="width:103px;">14203</td>
			<td>superparamagnetic beads&#160;</td>
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			<td height="21" style="height:21px;width:231px;">Fibronectin&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">F1141-5MG</td>
			<td>Fibronectin from bovine plasma</td>
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			<td height="21" style="height:21px;width:231px;">Apo-Transferrin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">T1428-50MG</td>
			<td>Bovine Apo-Transferrin</td>
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			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">A7906-500G</td>
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			<td height="42" style="height:42px;width:231px;">DMEM high glucose, GlutaMAX supplement, pyruvate&#160;</td>
			<td style="width:115px;">Life Technologies</td>
			<td style="width:103px;">31966-021</td>
			<td>DMEM+GlutaMAX-I 500 ml&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:231px;">60*15 mm culture dish</td>
			<td style="width:115px;">Falcon</td>
			<td style="width:103px;">353004</td>
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analyzing cell surface adhesion remodeling in response to mechanical tension using magnetic beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

The schematic of the technique is illustrated in Figure 1a. Following ligand conjugation, magnetic beads are incubated with cells for 20 min, and then a permanent magnet is used to apply tensile forces of about 30-40 pN for various amount of time. Figure 1b shows 2.8 &#181;m FN-coated magnetic beads bound to MRC5 cell adhesion receptors.
The wash steps of superparamagnetic beads after cell lysis are crucial and determine the degree of purification. A minimum o...

Watch this Scientific Journal Video about Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads at JoVE.com

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

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8.4K Views.  Centre de recherche UGA - INSERM U1209 - CNRS UMR. Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

Video: Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...