Microfluidic Device to Study Phosphoinositide Gradients in Model Membranes

Abstract

The availability of phosphoinositide-(3,4,5)-trisphosphate (PI(3,4,5)P3) in the plasma membrane (PM) dictates downstream activation of cellular division, growth and motility via the PI3K-Akt pathway. PI(3,4,5)P3 composition in the PM is regulated by the competition of PI3K kinase versus the phosphatase PTEN, whose role is to dephosphorylate PI(3,4,5)P3 to produce PI(4,5)P2. These opposing actions in motile cells result in a gradient of PI(3,4,5)P3 at the leading edge giving way to PI(4,5)P2 at the trailing. Despite this important feature in cells like Dictyostelium and neutrophils, there are no current model systems available to study gradients of phosphoinositides. The work presented here shows a novel methodology using microfluidics to generate PI(4,5)P2 gradients in supported lipid bilayers (SLB).We have successfully developed a tapered microfluidic device where a gradient of PI(4,5)P2 can be generated with both temporal and spatial control. The tapered design allows for a choice of channel width to observe the gradient ranging from 500 μm to 50 μm. Using total internal reflectance fluorescence (TIRF) microscopy we can quantify single molecule dynamics in the channel. In addition to this, we demonstrate Ca2+ gradient dependent domain formation in SLBs composed of PI(4,5)P2, PI(4,5)P2 and cholesterol, and well as POPS containing membranes.This novel method will allow for the investigation of gradients in membranes that where not attainable before. The advantage of having a choice to work with a model system in addition to the cellular systems is the ability to test hypotheses outside the complex cellular milieu to see if a trend is preserved. With this device we hope to start piecing together the most basic components necessary to maintain or degrade cellular gradients whose results could continue to answer the ultimate question of how lipid organization affects the information transduction within a cell.