Unrestricted diffusion of exogenous and endogenous PIP2 in baby hamster kidney and Chinese hamster ovary cell plasmalemma

Alp Yaradanakul, Donald W. Hilgemann

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

We used two approaches to characterize the lateral mobility of phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasmalemma of baby hamster kidney and Chinese hamster ovary fibroblasts. First, nitrobenzoxadiazole-labeled C6-phosphatidylcholine and C16-PIP2 were incorporated into plasma membrane "lawns" (∼20 × 30 μm) from these cells and into the outer monolayer of intact cells. Diffusion coefficients determined by fluorescence recovery after photobleaching were similar for the two lipids and were higher in lawns, ∼0.3 μm 2/s, than on the cell surface, ∼0.1 μm2/s. For membrane lawns, the fractional recoveries (75-90%) were close to those expected from the fraction of total membrane bleached, and labeling by the probes was several times greater than for intact cells. Second, we analyzed cells expressing M1 muscarinic receptors and green fluorescent protein fused with PIP2-binding pleckstrin-homology domains, Tubby domains or diacylglycerol (DAG)-binding C1 domains. On-cell gigaseal patches were formed with pipette tips >5 μm in diameter. When the agonist carbachol (0.3 mm) was applied either within or outside of the pipette, lipid signals crossed the pipette barrier rapidly in both directions and membrane blebbing occurred on both membrane sides. Accurate simulations of lipid gradients required diffusion coefficients >1 μm2/s. Exogenous DAG also crossed the pipette barrier rapidly. In summary, we found no evidence for restricted diffusion of signaling lipids in these cells. The lower mobility and incorporation of phospholipid at the extracellular leaflet may reflect a more ordered and condensed extracellular monolayer, as expected from previous studies.

Original languageEnglish (US)
Pages (from-to)53-67
Number of pages15
JournalJournal of Membrane Biology
Volume220
Issue number1-3
DOIs
StatePublished - Dec 2007

Keywords

  • Diacylglycerol
  • Fluorescence recovery after photobleaching
  • Membrane raft
  • Phosphatidylinositol
  • Phospholipid diffusion
  • Plasmalemma

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

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