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

Summary

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.

Abstract

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-δ1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) interaction as an example. The apparent dissociation constant (K_d,app) for this interaction was 0.39 ± 0.05 µM, similar to K_d,app values obtained by others. As previously observed, the PLC-δ1 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's capacity to interact with membranes.

Introduction

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 others^1,2,3,4. Dynamic phosphorylation of the inositol head gro....

Protocol

1. Cleaning the Glass Coverslips

1. 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 °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-butoxyethoxy)ethanol, sodium 1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate and non-hazardous additions.
2. Using tweezers, arrange the coverslips (40 mm len....

Results

We used the pH modulation assay to study the PLC-δ1 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 of a.......

Discussion

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 activation^6,7. Due to their role in cellular physi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Coverslip
Glass Coverslips: Rectangles	Fisher Scientific	12-544B	22 x 40 x 0.16 - 0.19 mm, No. 1 1/2; Borosilicate Glass
7X Cleaning Solution	MP Biomedicals	976670	Detergent
PYREX Crystallizing Dish	Corning	3140-190	Borosilicate glass dish with a flat bottom; Diameter x Height (190 x 100 mm); Distributor: VWR (89090-700)
Sentry Xpress 2.0	Paragon Industries	SC-2	Kiln
Name	Company	Catalog Number	Comments
PDMS
Sylgard 184 Silicone Elastomer Kit	Dow Corning	4019862	Polydimethylsiloxane (PDMS); Distributor: Ellsworth Adhesives
PYREX Desiccator	VWR	89134-402	Vacuum Rated
Biopsy punch	Harris	15110-10	Harris Uni-Core; 1.0 mm diameter; Miltex Biopsy Punch with Plunger (Cat. No. 15110-10) can be used as an alternative
Name	Company	Catalog Number	Comments
Device
Plasma Cleaning System	PlasmaEtch	PE25-JW	2-stage Direct Drive Oil Vacuum Pump, O2 service (Krytox Charged)
Digital Hot Plate	Benchmark	H3760-H	Purchased through Denville Scientific (Cat. No. 1005640)
Frosted Micro Slides	VWR	48312-003	Frosted, Selected, and Precleaned; Made of Swiss Glass; Thickness: 1 mm; Dimensions: 75 x 25 mm; GR 144
Name	Company	Catalog Number	Comments
Mold
AutoCAD	Autodesk	v.2016	Drafting software for the photomask design
Photomask	CAD/Art Services	N/A	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
Silicone Wafers	University Wafer	1575	Prime Grade, Single Side Polished; 100 mm (4 inch) Diameter; 525 um Thickness
SU-8 50	MicroChem Corp.	N/A	Negative Tone Photoresist; Penn State Nanofabrication Facility Property
SU-8 Developer	MicroChem Corp.	N/A	Penn State Nanofabrication Facility Property
Name	Company	Catalog Number	Comments
SUV
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine	Avanti Polar Lipids	850457C	POPC
L-α-phosphatidylinositol-4-phosphate	Avanti Polar Lipids	840045X	PI4P
L-α-phosphatidylinositol-4,5-bisphosphate	Avanti Polar Lipids	840046X	PI(4,5)P2
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine	Avanti Polar Lipids	850757C	POPE; Required for the synthesis of oSRB-POPE
Lissamine Rhodamine B Sulfonyl Chloride (mixed isomers)	ThermoFisher Scientific	L-20	Required for the synthesis of oSRB-POPE
pH Sensitive Fluorescent Lipid Probe (oSRB-POPE)	In-house	N/A	In-house Synthesis (Huang D. et al. 2013)
Glass Scintillation Vial	VWR	66022-065	20 mL volume capacity
Aquasonic 250D	VWR	N/A	Ultrasonic Water Bath
Nuclepore Track-Etched Membranes	Whatman	110605	Polycarbonate Membrane; Diameter: 25 mm; Pore Size: 0.1 um; Distributor: Sigma-Aldrich
Chloroform	VWR	CX1054-6	HPLC grade
LIPEX Extruder	Transferra Nanosciences	T.001	LIPEX 10 mL Thermobarrel Extruder
Viscotek 802 DLS	Malvern Instruments	N/A	Dynamic Light Scattering; Penn State X-Ray Crystallography Facility Property
Name	Company	Catalog Number	Comments
Data Analysis
GraphPad Prism	GraphPad Software	v.6	Curve-fitting software for data analysis
Name	Company	Catalog Number	Comments
Microscope
Axiovert 200M Epifluorescence Microscope	Carl Zeiss Microscopy	N/A	Microscope
AxioCam MRm Camera	Carl Zeiss Microscopy	N/A	Camera
X-Cite 120	Excelitas Technologies	N/A	Light Source
Alexa 568 Filter Set	Carl Zeiss Microscopy	N/A	Ex/Em 576/603 nm
AxioVision LE64 v.4.9.1.0 Software	Carl Zeiss Microscopy	N/A	Image Processing Software
Name	Company	Catalog Number	Comments
Other
Tips	VWR	10034-132	200 uL pipette tips; Thin and smooth tip for applying the protein solution into the microfluidic channel
Tips	VWR	53509-070	10 uL pipette tips; Thin and smooth tip for applying the vesicle solution into the microfluidic channel
Orion Star A321 pH meter	Thermo Scientific	STARA3210	pH meter
Orion micro pH probe	Thermo Scientific	8220BNWP	micro pH probe
N-(2-Hydroxyethyl)-Piperazine-N'-(2-Ethanesulfonic Acid)	VWR	VWRB30487	HEPES, Free Acid
Sodium Chloride	VWR	BDH8014-2.5KGR	NaCl
Tubing	Allied Wire & Cable	TFT-200-24 N	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
Nitrogen Gas - Industrial	Praxair	N/A	Local Provider
Oxygen Gas - Industrial	Praxair	N/A	Local Provider
Liquid Nitrogen	Praxair	N/A	Local Provider