Nanoscale Landscape of Phosphoinositides Revealed by Specific Pleckstrin Homology (PH) Domains Using Single-molecule Superresolution Imaging in the Plasma Membrane

Chen Ji,Yongdeng Zhang,Pingyong Xu,Tao Xu,Xuelin Lou

doi:10.1074/jbc.m115.663013

Abstract

Both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are independent plasma membrane (PM) determinant lipids that are essential for multiple cellular functions. However, their nanoscale spatial organization in the PM remains elusive. Using single-molecule superresolution microscopy and new photoactivatable fluorescence probes on the basis of pleckstrin homology domains that specifically recognize phosphatidylinositides in insulin-secreting INS-1 cells, we report that the PI(4,5)P2 probes exhibited a remarkably uniform distribution in the major regions of the PM, with some sparse PI(4,5)P2-enriched membrane patches/domains of diverse sizes (383 ± 14 nm on average). Quantitative analysis revealed a modest concentration gradient that was much less steep than previously thought, and no densely packed PI(4,5)P2 nanodomains were observed. Live-cell superresolution imaging further demonstrated the dynamic structural changes of those domains in the flat PM and membrane protrusions. PI4P and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) showed similar spatial distributions as PI(4,5)P2. These data reveal the nanoscale landscape of key inositol phospholipids in the native PM and imply a framework for local cellular signaling and lipid-protein interactions at a nanometer scale.

Highlights

Phosphatidylinositides in the plasma membrane (PM) are pivotal for cellular functions
Both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) are independent plasma membrane (PM) determinant lipids that are essential for multiple cellular functions
Using single-molecule superresolution microscopy and new photoactivatable fluorescence probes on the basis of pleckstrin homology domains that recognize phosphatidylinositides in insulin-secreting INS-1 cells, we report that the PI[4,5]P2 probes exhibited a remarkably uniform distribution in the major regions of the PM, with some sparse PI[4,5]P2-enriched membrane patches/domains of diverse sizes (383 ؎ 14 nm on average)

Summary

Introduction

Phosphatidylinositides in the plasma membrane (PM) are pivotal for cellular functions. Significance: This result may imply a new working model of phosphatidylinositides at nanometer scale Both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) are independent plasma membrane (PM) determinant lipids that are essential for multiple cellular functions. Their nanoscale spatial organization in the PM remains elusive. PI4P and phosphatidylinositol [3,4,5]-trisphosphate (PI[3,4,5]P3) showed similar spatial distributions as PI[4,5]P2 These data reveal the nanoscale landscape of key inositol phospholipids in the native PM and imply a framework for local cellular signaling and lipid-protein interactions at a nanometer scale

Methods

Results

Conclusion