Our work has been supported by grants from American Heart Association (Greater Southeast Affiliate) Beginning-grant-in-aid 0765185B, the Elsa U. Pardee Foundation research grant, and Wayne State University intramural startup fund and Cardiovascular Research Institute Isis Initiative award. This method of in vitro CFTR macromolecular complex assembly was originally pioneered by Dr. A.P. Naren (University of Tennessee Health Science Center).

1. Expression and Purification of Recombinant Tagged Fusion Proteins in Bacteria
<ol>
	<li>Amplify defined regions of the C-tails (the last 50-100 amino acids containing the PDZ motifs at C-terminus) for CFTR, LPA2, MRP2, MRP4, &beta;2AR, and NHERFs (full-length or PDZ1 or PDZ2 domains) by PCR approach.</li>
	<li>Clone the PCR products into pGEX4T-1 vector for GST-fusion proteins (such as GST-NHERFs, GST-MRP4 CT), pMAL-C2 vector for MBP-fusion proteins (such as MBP-&beta;2AR CT, MBP-CFTR CT), and pET30 for His-S-fusion proteins (such as His-S-CFTR CT, His-S-PDZK1).</li>
	<li>Transform in a protease-deficient E. coli strain (BL21-DE3) to minimize possible recombinant protein degradation.</li>
	<li>Grow the culture overnight at 37 &deg;C in Luria-Bertani medium (pH 7.0) containing appropriate antibiotics (such as ampicillin or kanamycin). Dilute the overnight culture at 1:10 and grow for another 2 hr at 37 &deg;C. Induce with 0.5-1 mM IPTG for the next 4 hr at 30 &deg;C. Pellet the cells by centrifugation at 8,000 &times; g for 10 min at 4 &deg;C.</li>
	<li>Lyse the cells in Sucrose Buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM PMSF, and 10% sucrose) containing lysozyme (1 mg/ml), 0.2% Triton X-100, and protease inhibitors. Use 20 ml for cell pellet originating from 1 L of culture.</li>
	<li>Mix on a rotary shaker for 30 min at 4 &deg;C.</li>
	<li>Spin at 20,000 &times; g for 30 min at 4 &deg;C. Collect the clear supernatant.</li>
	<li>Into the clear supernatant, add 1 ml of the following resin/agarose beads (50% slurry in Sucrose Buffer):
	<ul>
		<li>Glutathione agarose beads for GST fusion proteins.</li>
		<li>Talon beads for His-S fusion proteins.</li>
		<li>Amylose resin for MBP fusion proteins.</li>
	</ul>
	</li>
	<li>Mix for 4 hr at 4 &deg;C on a rotary shaker.</li>
	<li>Wash the beads by resuspending in 1x PBS (15 ml), mixing for 2 min, spinning at 800 &times; g for 2 min, and discarding the supernatant. Repeat this step for 6 times.</li>
	<li>Elute the protein from the beads by using respective elution buffer (2 ml elution buffer per 1 ml of beads).
	<ul>
		<li>Elution buffer for GST fusion proteins: 25 mM Tris-HCl (pH 8.0), 140 mM NaCl, and 20 mM reduced glutathione.</li>
		<li>Elution buffer for His fusion proteins: 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 200 mM imidazole.</li>
		<li>Elution buffer for MBP fusion proteins: 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, 1 mM DTT, and 10 mM maltose.</li>
	</ul>
	</li>
	<li>Dialyze the eluted proteins against 2 L of 1x PBS at 4 &deg;C (to avoid possible protein degradation). Change PBS every 4 hr for 4 times. Concentrate the protein using a Centricon filter (10,000 MW cutoff, Millipore) and store as small aliquots at -80 &deg;C.</li>
	<li>Determine the protein concentration by the Bradford method. Assess protein quality by SDS-PAGE using BSA as standards. If the integrity of the protein is not satisfactory, secondary purification procedures such as gel filtration or ion exchange may be used. (e.g., we have used a G-75 Sepharose column to further purify GST-fusion protein).</li>
</ol>
2. Cell Culture and Cell Lysate Preparation
<ol>
	<li>Culture baby hamster kidney (BHK) cells that stably over-express CFTR-wt and CFTR-his10 or BHK cells that transiently over-express Flag-LPA2-wt or Flag-LPA2-&Delta;STL, and Madin-Darby canine kidney (MDCK) cells that stably over-express MRP2 in Eagle's minimum essential medium (MEM) containing 10% fetal calf serum and penicillin/streptomycin in polystyrene flasks at 37 &deg;C with 5% CO2.</li>
	<li>Culture human embryonic kidney 293 (HEK293) cells that stably over-express CFTR-wt and CFTR-his10 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin in polystyrene flasks at 37 &deg;C with 5% CO2.</li>
	<li>Lyse the cells in Lysis Buffer (PBS - 0.2% Triton-X-100 supplemented with a mixture of protease inhibitors containing 1 mM phenylmethylsulphonyl fluoride, 1 &mu;g/ml aprotinin, 1 &mu;g/ml leupeptin, 1 &mu;g/ml pepstatin); use 500 &mu;l lysis buffer for each 60-mm Petri dish, and 1000 &mu;l lysis buffer for each 100-mm Petri dish.</li>
	<li>Rock the cell lysates for 20 min at 4 &deg;C, and remove the insoluble material by centrifugation at 16,000 &times; g for 15 min at 4 &deg;C. Determine the protein concentration by the Bradford assay.</li>
</ol>
3. In Vitro Assembly of a CFTR-containing Macromolecular Complex (CFTR-PDZK1-MRP4)
<ol>
	<li>Into 200 &mu;l of lysis buffer (PBS - 0.2% Triton-X 100 + protease inhibitors), add 20 &mu;g purified GST-MRP4-C terminal 50 a.a. (CT50) fusion protein.</li>
	<li>Add various amounts (0-40 &mu;g) of purified His-S-PDZK1 fusion protein.</li>
	<li>Mix the two proteins on a rotary mixer at 22 &deg;C for 1-2 hr.</li>
	<li>Add 20 &mu;l glutathione beads (50% slurry) into the protein mixture, and continue to mix for another 1 hr. This step is also referred to as pair-wise binding.</li>
	<li>During this time, prepare the HEK293 cell lysates that overexpress Flag-CFTR (wt) as described above in Step 2).</li>
	<li>Wash the complex twice with lysis buffer. Spin at 800 &times; g for 1 min for each wash and carefully aspirate the supernatant after each wash. Use caution not to suck off the beads in the bottom.</li>
	<li>Add the above-prepared HEK293 cell lysates to the beads, and gently mix at 4 &deg;C for 3 hr (or overnight).</li>
	<li>Wash the beads extensively 3 times with lysis buffer as described in Step 3.6).</li>
	<li>Elute the proteins using 30 &mu;l Sample Buffer (5&times;): 0.6 M Tris-HCl (pH 6.8), 50% glycerol, 2% SDS, and 0.1% bromophenol blue; containing 5% &beta;-mercaptoethanol.</li>
	<li>Incubate in 37 &deg;C water bath for 10-15 min, and spin at 5,000 &times; g for 30 s to precipitate the beads.</li>
	<li>Load all the eluate onto 4-15% gel.</li>
	<li>Run SDS-PAGE for about 30-40 min.</li>
	<li>Transfer protein bands to the PVDF membrane, for 1.5 hr.</li>
	<li>Block the membrane in TBS-Tween containing 5% non fat milk.</li>
	<li>Incubate the membrane with primary antibody (such as anti-CFTR IgG, R1104) at 4 &deg;C, overnight.</li>
	<li>Wash the membrane with TBS-Tween for 5 min, 6 times.</li>
	<li>Incubate the membrane with secondary antibody (such as goat anti-mouse HRP-conjugated 2nd antibody) for 45 min at 22 &deg;C.</li>
	<li>Wash the membrane with TBS-Tween for 5 min, 6 times.</li>
	<li>Visualize the protein bands via ECL.</li>
	<li>Representative data are shown in Figure 1.</li>
</ol>
4. Representative Results
An example of CFTR-containing macromolecular signaling complex that was assembled in vitro is shown in Figure 1. A macromolecular complex was formed between MRP4 C-terminal 50 amino acids (MRP4-CT50), PDZK1, and full-length CFTR (Figure 1, bottom). The complex formation increased dose-dependently with increasing amounts of the intermediary protein, PDZK1 (Figure 1, bottom)18.
<img alt="Figure 1" src="/files/ftp_upload/4091/4091fig1.jpg" /> 
Figure 1. A pictorial representation of the macromolecular complex assay (top). A macromolecular complex was detected in vitro with three proteins (GST-MRP4-CT50, His-S-PDZK1, and Flag-CFTRwt) in a dose-dependent manner (bottom)18.
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>CFTR-NHERF1-&beta;2AR (ref.14)</td>
			<td>CFTR-NHERF2-LPA2 (ref. 15)</td>
			<td>CFTR-PDZ proteins-MRP2 (ref. 17)</td>
			<td>CFTR-PDZK1-MRP4 (ref. 16)</td>
		</tr>
		<tr>
			<td>Affinity beads</td>
			<td>Amylose resin</td>
			<td>S-protein agarose</td>
			<td>Amylose resin</td>
			<td>Glutathione agarose</td>
		</tr>
		<tr>
			<td>Purified protein-1</td>
			<td>MBP-&beta;2AR CT</td>
			<td>His-S-CFTR CT</td>
			<td>MBP-CFTR CT</td>
			<td>GST-MRP4 CT</td>
		</tr>
		<tr>
			<td>Purified protein-2</td>
			<td>GST-NHERF1</td>
			<td>GST-NHERF2</td>
			<td>GST-PDZ proteins</td>
			<td>His-S-PDZK1</td>
		</tr>
		<tr>
			<td>Purified protein-3 (or cell lysates)</td>
			<td>CFTR-wt or CFTR-his10 (BHK or HEK cell lysates)</td>
			<td>Flag-LPA2-wt or Flag-LPA2-&Delta;STL (BHK cell lysates)</td>
			<td>MRP2 (MDCK cell lysates)</td>
			<td>Purified Flag-CFTR-wt or CFTR-his10 (or cell lysates)</td>
		</tr>
		<tr>
			<td>Antibody</td>
			<td>Anti-CFTR IgG</td>
			<td>Anti-Flag HRP</td>
			<td>Anti-MRP2 IgG</td>
			<td>Anti-Flag HRP</td>
		</tr>
	</tbody>
</table>
Table 1. Summary of various CFTR-containing macromolecular complexes assembled in vitro.

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No conflicts of interest declared.

In this protocol we demonstrated a method for in vitro assembling and detection of a CFTR containing macromolecular signaling complex using purified proteins (or protein fragments) and/or cell lysates as reported previously16-19,29,30. To achieve best results the following critical points during the preparation process require special attention:
<ul>
	<li>It is important that the pH of the elution buffer be adjusted to 8.0 after adding reduced glutathione when purifying the GST-fusion protein as d...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>pGEX4T-1 vector</td>
			<td>GE Healthcare</td>
			<td>28-9545-49</td>
			<td>formerly Amersham Biosciences</td>
		</tr>
		<tr>
			<td>pMAL-C2 vector</td>
			<td>New England BioLabs</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>pET30 vector</td>
			<td>EMD Chemicals</td>
			<td>69077-3</td>
			<td>formerly Novagen</td>
		</tr>
		<tr>
			<td>Glutathione agarose beads</td>
			<td>BD Biosciences</td>
			<td>554780</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amylose resin</td>
			<td>New England BioLabs</td>
			<td>E8021S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Talon beads</td>
			<td>Clontech</td>
			<td>635501</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>reduced glutathione</td>
			<td>BD Biosciences</td>
			<td>554782</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>imidazole</td>
			<td>Fisher</td>
			<td>BP305-50</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>maltose</td>
			<td>Fisher</td>
			<td>BP684-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>S-protein agarose</td>
			<td>EMD Chemicals</td>
			<td>69704-3</td>
			<td>formerly Novagen</td>
		</tr>
		<tr>
			<td>Anti-Flag HRP</td>
			<td>Sigma</td>
			<td>A8592</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CFTR IgG</td>
			<td>Custom-made</td>
			<td>R1104</td>
			<td>mAb recognizing CFTR epitope at a.a. 722-734</td>
		</tr>
		<tr>
			<td>Anti-MRP2 IgG</td>
			<td>Chemicon International</td>
			<td>MAB4148</td>
			<td>Now a part of Millipore</td>
		</tr>
	</tbody>
</table>
Table 2. Specific reagents and equipment.

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

Watch this Scientific Journal Video about In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes at JoVE.com

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

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Biology

10.3K Views.  Wayne State University School of Medicine. סיסטיק פיברוזיס הטרנסממברני הרגולטור המוליכות (CFTR), ערוץ כלוריד אפיתל, דווחה אינטראקציה עם חלבונים שונים לווסת תהליכים תאיים חשובים, ביניהם את PDZ CFTR מוטיב בתיווך אינטראקציות תועדו היטב. פרוטוקול זה מתאר שיטות שפיתחנו כדי להרכיב macromolecular PDZ תלוי CFTR איתות מורכבת

Video: In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.