Name: pip-on-a-chip: a label-free study of protein-phosphoinositide interactions
Uploaded: 2017-07-27T13:00:00.000+00:00
Duration: 10 min 58 s
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).

The authors have nothing to disclose.

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.

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No. 1005640)</td></tr><tr><td>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><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Mold&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AutoCAD</td><td>Autodesk</td><td>v.2016</td><td>Drafting software for the photomask design</td></tr><tr><td>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><td>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><td>SU-8 50</td><td>MicroChem Corp.</td><td>N/A</td><td>Negative Tone Photoresist; Penn State Nanofabrication Facility Property</td></tr><tr><td>SU-8 Developer</td><td>MicroChem Corp.</td><td>N/A</td><td>Penn State Nanofabrication Facility Property</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;SUV&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine</td><td>Avanti Polar Lipids</td><td>850457C</td><td>POPC</td></tr><tr><td>L-&amp;alpha;-phosphatidylinositol-4-phosphate</td><td>Avanti Polar Lipids</td><td>840045X</td><td>PI4P</td></tr><tr><td>L-&amp;alpha;-phosphatidylinositol-4,5-bisphosphate</td><td>Avanti Polar Lipids</td><td>840046X</td><td>PI(4,5)P2</td></tr><tr><td>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><td>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><td>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><td>Glass Scintillation Vial</td><td>VWR</td><td>66022-065</td><td>20 mL volume capacity</td></tr><tr><td>Aquasonic 250D</td><td>VWR</td><td>N/A</td><td>Ultrasonic Water Bath</td></tr><tr><td>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><td>Chloroform</td><td>VWR</td><td>CX1054-6</td><td>HPLC grade</td></tr><tr><td>LIPEX Extruder</td><td>Transferra Nanosciences</td><td>T.001</td><td>LIPEX 10 mL Thermobarrel Extruder</td></tr><tr><td>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><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Data Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GraphPad Prism</td><td>GraphPad Software</td><td>v.6</td><td>Curve-fitting software for data analysis</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscope&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Axiovert 200M Epifluorescence Microscope</td><td>Carl Zeiss Microscopy</td><td>N/A</td><td>Microscope</td></tr><tr><td>AxioCam MRm Camera</td><td>Carl Zeiss Microscopy</td><td>N/A</td><td>Camera</td></tr><tr><td>X-Cite 120</td><td>Excelitas Technologies</td><td>N/A</td><td>Light Source</td></tr><tr><td>Alexa 568 Filter Set</td><td>Carl Zeiss Microscopy</td><td>N/A</td><td>Ex/Em 576/603 nm</td></tr><tr><td>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><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>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><td>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><td>Orion Star A321 pH meter</td><td>Thermo Scientific</td><td>STARA3210</td><td>pH meter</td></tr><tr><td>Orion micro pH probe</td><td>Thermo Scientific</td><td>8220BNWP</td><td>micro pH probe</td></tr><tr><td>N-(2-Hydroxyethyl)-Piperazine-N&#039;-(2-Ethanesulfonic Acid)</td><td>VWR</td><td>VWRB30487</td><td>HEPES, Free Acid</td></tr><tr><td>Sodium Chloride</td><td>VWR</td><td>BDH8014-2.5KGR</td><td>NaCl</td></tr><tr><td>Tubing</td><td>Allied Wire &amp;amp; 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><td>Nitrogen Gas - Industrial</td><td>Praxair</td><td>N/A</td><td>Local Provider</td></tr><tr><td>Oxygen Gas - Industrial</td><td>Praxair</td><td>N/A</td><td>Local Provider</td></tr><tr><td>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 ...

pip-on-chip-label-free-study-protein-phosphoinositide

Research

JoVE Journal

Bioengineering

9.4K Views.  The Pennsylvania State University. 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.

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

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

Biochemistry

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Chemistry

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

pH Modulation Assay on Supported Lipid Bilayers to Detect Protein-Phosphoinositide Interactions

Source: Shengjuler, D., et al., <a href="https://app.jove.com/v/55869">PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions.</a> J. Vis. Exp. (2017)
In this video, we demonstrate protein-phosphoinositide interactions in a microfluidic pattern using the pH modulation assay, using pH-sensitive fluorescent dye tagged to phosphatidylethanolamine lipid of a supported lipid bilayer, called a microfluidic platform. The modulations in pH after protein binding to phospholipid cause fluorescence quenching.

Source: Shengjuler, D., et al., PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions. J. Vis. Exp. (2017)
In this video, we ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-lipid interactions

Phosphoinositides and PIPs

Phosphoinositides are a group of phospholipids containing a glycerol backbone with two fatty acid chains and a phosphate attached to a myoinositol sugar ring. The inositol head group extends into the cytoplasm, where it is modified by adding phosphate groups to form phosphatidylinositol phosphates or PIPs.
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane compartments imparts &#8220;surface identity&#8221; to the cell membrane. The phosphorylated heads of PIPs are recognized and bound by particular proteins, including proteins that help in vesicle coat assembly.
For example, the cytosolic side of the plasma membrane has high concentrations of the PI(4,5)P2, which helps recruit proteins such as dynamin and AP2 for clathrin-mediated endocytosis. PI(4,5)P2 is rapidly formed and destroyed by localized enzymes at particular locations and at specific times. This helps control the precise timing of clathrin-coated vesicle formation. Once the coated vesicle pinches off the plasma membrane during endocytosis, PI(4,5)P2 is destroyed from the site.
Many such PIs are involved in the secretory or endocytic pathways, such as&#160; PI(3)P localized at early endosomes and intraluminal vesicles of late endosomes,&#160; PI(4)P localized at the TGN, secretory granules, and synaptic vesicles; and PI(3,5)P2 localized at the late endosome boundary membrane.

Phosphoinositides are a group of phospholipids containing a glycerol backbone with two fatty acid chains and a phosphate attached to a myoinositol sugar ...