Name: pip-on-a-chip: a label-free study of protein-phosphoinositide interactions
Uploaded: 2017-07-27T13:00:00
Description: Watch this Scientific Journal Video about PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions at JoVE.com

D.S. and C.E.C. were supported, in part, by grant AI053531 (NIAID, NIH); S.S and P.S.C. were supported by grant N00014-14-1-0792 (ONR).

1. Cleaning the Glass Coverslips
<ol>
	<li>Dilute 7x Cleaning Solution (see Materials Table) 7-fold with deionized water in a 100 mm deep borosilicate glass dish with a flat bottom and heat it up to 95 &#176;C on a level hot plate for 20 min or until the cloudy solution becomes clear. 
	NOTE: The solution will be hot, use caution to avoid bodily injuries. The 7x Cleaning Solution is a proprietary blend of hexasodium [oxido-[oxido(phosphonatooxy)phosphoryl]oxyphosphoryl] phosphate, 2-(2-butoxyethox...

Each PIP variant, albeit at low concentrations, is present on the cytosolic surface of specific organelles where they contribute to the establishment of a unique physical composition and functional specificity of the organellar membrane1. One of the most important uses of PIPs is as a specific docking platform for the multitude of proteins requiring specific subcellular localization and/or activation6,7. Due to their role in cellular physi...

Myriad interactions and biochemical processes take place on two-dimensionally fluid membrane surfaces. Membrane-enclosed organelles in eukaryotic cells are unique not only in biochemical processes and their associated proteome but also in their lipid composition. One exceptional class of phospholipids is phosphoinositides (PIPs). Even though they comprise only 1% of the cellular lipidome, they play a crucial role in signal transduction, autophagy, and membrane trafficking, among others1,2,3,4. Dynamic phosphorylation of the inositol head group by cellular PIP kinases gives rise to seven PIP headgroups that are mono-, bis-, or tris-phosphorylated5. Additionally, PIPs define the subcellular identity of membranes and serve as specialized membrane docking sites for proteins/enzymes containing one or more phosphoinositide-binding domains, for example, Pleckstrin Homology (PH), Phox Homology (PX), and epsin N-terminal Homology (ENTH)6,7. One of the best-studied PIP-binding domains is phospholipase C (PLC)-&#948;1 PH domain that specifically interacts with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) within a high nanomolar-low micromolar range affinity8,9,10,11.
A variety of qualitative and quantitative in vitro methods have been developed and used to study the mechanism, thermodynamics, and specificity of these interactions. Among the most commonly used PIP-binding assays are surface plasmon resonance (SPR), isothermal calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy, liposome flotation/sedimentation assay, and lipid-blots (Fat-blots/PIP-strips)12,13. Even though these are extensively utilized, they all have many disadvantages. For example, SPR, ITC, and NMR require large amounts of sample, expensive instrumentation, and/or trained personnel12,13. Some assay formats such as antibody-based lipid-blots utilize water soluble forms of PIPs and present them in a nonphysiological manner12,14,15,16. In addition, lipid-blots cannot be quantitated reliably and they have often resulted in false positive/negative observations12,17,18. To overcome these challenges and improve upon the current tool set, a new label-free method was established based on a supported lipid bilayer (SLB) in the context of a microfluidic platform, which was successfully applied to the study of protein-PIP interactions (Figure 1)19.
The strategy employed for detecting protein-PIP interactions is based on pH modulation sensing. This involves a pH-sensitive dye that has ortho-Sulforhodamine B (oSRB) directly conjugated to phosphatidylethanolamine lipid head group20. The oSRB-POPE probe (Figure 2A) is highly fluorescent at low pH and quenched at high pH with a pKa around 6.7 within 7.5 mol% PI(4,5)P2-containing SLBs (Figure 5B). PLC-&#948;1 PH domain has been used extensively for validating protein-PIP-binding methodologies due to its high specificity towards PI(4,5)P2 (Figure 5A)21,22,23,24,25.Hence, we reasoned that the PLC-&#948;1 PH domain can be used to test its binding to PI(4,5)P2 through the PIP-on-a-chip assay. The PH domain construct used in this study has a net positive charge (pI 8.4), and thus attracts OH- ions (Figure 5C). Upon binding to PI(4,5)P2-containing SLBs, the PH domain brings the OH- ions to the membrane surface, which in turn modulates the interfacial potential and shifts the protonation state of oSRB-POPE (Figure 5C)26. As a function of the PH domain concentration, the fluorescence is quenched (Figure 6A). Finally, the normalized data is fit to a binding isotherm to determine the affinity of the PH domain-PI(4,5)P2 interaction (Figure 6B, 6C).
In this study, a detailed protocol is provided to perform protein binding to PIP-containing SLBs within a microfluidic platform. This protocol takes the reader from assembling the microfluidic device and vesicle preparation to SLB formation and protein binding. In addition, directions for data analysis to extract affinity information for the PLC-&#948;1 PH domain-PI(4,5)P2 interaction are provided.

<table border="0" cellpadding="0" cellspacing="0" width="1638">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Coverslip</td>
			<td style="width:156px;"></td>
			<td style="width:123px;"></td>
			<td style="width:997px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Coverslips: Rectangles</td>
			<td>Fisher Scientific</td>
			<td>12-544B</td>
			<td>22 x 40 x 0.16 - 0.19 mm, No. 1 1/2; Borosilicate Glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7X Cleaning Solution</td>
			<td>MP Biomedicals</td>
			<td>976670</td>
			<td>Detergent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PYREX Crystallizing Dish</td>
			<td>Corning</td>
			<td>3140-190</td>
			<td>Borosilicate glass dish with a flat bottom; Diameter x Height (190 x 100 mm); Distributor: VWR (89090-700)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sentry Xpress 2.0</td>
			<td>Paragon Industries</td>
			<td>SC-2</td>
			<td>Kiln</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDMS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning&#160;</td>
			<td>4019862</td>
			<td>Polydimethylsiloxane (PDMS); Distributor: Ellsworth Adhesives</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PYREX Desiccator</td>
			<td>VWR</td>
			<td>89134-402</td>
			<td>Vacuum Rated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biopsy punch</td>
			<td>Harris</td>
			<td>15110-10</td>
			<td style="width:997px;">Harris Uni-Core; 1.0 mm diameter; Miltex Biopsy Punch with Plunger (Cat. No. 15110-10) can be used as an alternative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Device</td>
			<td></td>
			<td></td>
			<td style="width:997px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma Cleaning System</td>
			<td>PlasmaEtch</td>
			<td>PE25-JW</td>
			<td>2-stage Direct Drive Oil Vacuum Pump, O2 service (Krytox Charged)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital Hot Plate</td>
			<td>Benchmark</td>
			<td>H3760-H</td>
			<td>Purchased through Denville Scientific (Cat. No. 1005640)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Frosted Micro Slides</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>Frosted, Selected, and Precleaned; Made of Swiss Glass; Thickness: 1 mm; Dimensions: 75 x 25 mm; GR 144</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Mold</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AutoCAD</td>
			<td>Autodesk</td>
			<td>v.2016</td>
			<td>Drafting software for the photomask design</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photomask</td>
			<td>CAD/Art Services</td>
			<td>N/A</td>
			<td>Design with black background and clear features was printed at 20k dpi resolution on a transparent mask (5 x 7 in) by CAD/Art Services</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicone Wafers</td>
			<td>University Wafer</td>
			<td>1575</td>
			<td>Prime Grade, Single Side Polished; 100 mm (4 inch) Diameter; 525 um Thickness</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 50</td>
			<td>MicroChem Corp.</td>
			<td>N/A</td>
			<td>Negative Tone Photoresist; Penn State Nanofabrication Facility Property</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SU-8 Developer</td>
			<td>MicroChem Corp.</td>
			<td>N/A</td>
			<td>Penn State Nanofabrication Facility Property</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SUV</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine</td>
			<td>Avanti Polar Lipids</td>
			<td>850457C</td>
			<td>POPC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-&#945;-phosphatidylinositol-4-phosphate</td>
			<td>Avanti Polar Lipids</td>
			<td>840045X</td>
			<td>PI4P</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-&#945;-phosphatidylinositol-4,5-bisphosphate&#160;</td>
			<td>Avanti Polar Lipids</td>
			<td>840046X</td>
			<td>PI(4,5)P2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine</td>
			<td>Avanti Polar Lipids</td>
			<td>850757C</td>
			<td>POPE; Required for the synthesis of oSRB-POPE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lissamine Rhodamine B Sulfonyl Chloride (mixed isomers)</td>
			<td>ThermoFisher Scientific</td>
			<td>L-20</td>
			<td>Required for the synthesis of oSRB-POPE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH Sensitive Fluorescent Lipid Probe (oSRB-POPE)</td>
			<td>In-house</td>
			<td>N/A</td>
			<td>In-house Synthesis (Huang D. et al. 2013)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Scintillation Vial</td>
			<td>VWR</td>
			<td>66022-065</td>
			<td>20 mL volume capacity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aquasonic 250D</td>
			<td>VWR</td>
			<td>N/A</td>
			<td>Ultrasonic Water Bath</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclepore Track-Etched Membranes</td>
			<td>Whatman</td>
			<td>110605</td>
			<td>Polycarbonate Membrane; Diameter: 25 mm; Pore Size: 0.1 um; Distributor: Sigma-Aldrich</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform</td>
			<td>VWR</td>
			<td>CX1054-6</td>
			<td>HPLC grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LIPEX Extruder</td>
			<td>Transferra Nanosciences</td>
			<td>T.001</td>
			<td>LIPEX 10 mL Thermobarrel Extruder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Viscotek 802 DLS</td>
			<td>Malvern Instruments</td>
			<td>N/A</td>
			<td>Dynamic Light Scattering; Penn State X-Ray Crystallography Facility Property</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Data Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism</td>
			<td>GraphPad Software</td>
			<td>v.6</td>
			<td>Curve-fitting software for data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Axiovert 200M Epifluorescence Microscope</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AxioCam MRm Camera</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Camera</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">X-Cite 120</td>
			<td>Excelitas Technologies</td>
			<td>N/A</td>
			<td>Light Source</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa 568 Filter Set</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Ex/Em 576/603 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AxioVision LE64 v.4.9.1.0 Software</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Image Processing Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tips</td>
			<td>VWR</td>
			<td>10034-132</td>
			<td>200 uL pipette tips; Thin and smooth tip for applying the protein solution into the microfluidic channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tips</td>
			<td>VWR</td>
			<td>53509-070</td>
			<td>10 uL pipette tips; Thin and smooth tip for applying the vesicle solution into the microfluidic channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Orion Star A321 pH meter</td>
			<td>Thermo Scientific</td>
			<td>STARA3210</td>
			<td>pH meter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Orion micro pH probe</td>
			<td>Thermo Scientific</td>
			<td>8220BNWP</td>
			<td>micro pH probe</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-(2-Hydroxyethyl)-Piperazine-N&#39;-(2-Ethanesulfonic Acid)</td>
			<td>VWR</td>
			<td>VWRB30487</td>
			<td>HEPES, Free Acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Chloride</td>
			<td>VWR</td>
			<td>BDH8014-2.5KGR</td>
			<td>NaCl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tubing</td>
			<td>Allied Wire &#38; Cable</td>
			<td>TFT-200-24 N</td>
			<td>Internal Diameter: 0.020-0.026 inches (0.051-0.066 cm); Wall Thickness: 0.010 inches (0.025 cm); Flexible Polytetrafluoroethylene Thin-Wall Tubing; Natural Color</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nitrogen Gas - Industrial</td>
			<td>Praxair</td>
			<td>N/A</td>
			<td>Local Provider</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oxygen Gas - Industrial</td>
			<td>Praxair</td>
			<td>N/A</td>
			<td>Local Provider</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid Nitrogen</td>
			<td>Praxair</td>
			<td>N/A</td>
			<td>Local Provider</td>
		</tr>
	</tbody>
</table>

pip-on-a-chip: a label-free study of protein-phosphoinositide interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

We used the pH modulation assay to study the PLC-&#948;1 PH domain-PI(4,5)P2 interaction within a PIP-on-a-chip microdevice (Figure 1). Through a detailed protocol, we demonstrated how to prepare and assemble microfluidic device components, make small unilamellar vesicles (SUVs) (Figure 2), form SLBs within a device (Figure 3), and tested protein binding to PIP-containing SLBs. A flowchart...

Watch this Scientific Journal Video about PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions at JoVE.com

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

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