Dynamic Meso-Scale Anchorage of GPI-Anchored Receptors in the Plasma Membrane: Prion Protein vs. Thy1

Yuri L Nemoto,Takahiro K Fujiwara,Rinshi S Kasai,Akihiro Kusumi,Taka A Tsunoyama,Hiroko Hijikata,Roger J Morris,Akihiro C E Shibata

doi:10.1007/s12013-017-0808-3

Abstract

The central mechanism for the transmission of the prion protein misfolding is the structural conversion of the normal cellular prion protein to the pathogenic misfolded prion protein, by the interaction with misfolded prion protein. This process might be enhanced due to the homo-dimerization/oligomerization of normal prion protein. However, the behaviors of normal prion protein in the plasma membrane have remained largely unknown. Here, using single fluorescent-molecule imaging, we found that both prion protein and Thy1, a control glycosylphosphatidylinositol-anchored protein, exhibited very similar intermittent transient immobilizations lasting for a few seconds within an area of 24.2 and 3.5 nm in diameter in CHO-K1 and hippocampal neurons cultured for 1- and 2-weeks, respectively. Prion protein molecules were immobile during 72% of the time, approximately 1.4× more than Thy1, due to prion protein’s higher immobilization frequency. When mobile, prion protein diffused 1.7× slower than Thy1. Prion protein’s slower diffusion might be caused by its transient interaction with other prion protein molecules, whereas its brief immobilization might be due to temporary association with prion protein clusters. Prion protein molecules might be newly recruited to prion protein clusters all the time, and simultaneously, prion protein molecules in the cluster might be departing continuously. Such dynamic interactions of normal prion protein molecules would strongly enhance the spreading of misfolded prion protein.

Highlights

In recent years, various membrane proteins and lipids in the plasma membrane (PM) have been found to undergo temporary entrapment or immobilization in meso-scale domains [1,2,3,4,5,6,7,8]
Suzuki et al [1, 2] reported that upon the engagement or colloidal goldinduced cross-linking of physiological levels of CD59, a glycosylphosphatidylinositol-anchored receptor (GPI-AR), CD59 clusters containing an average of about five CD59 molecules underwent alternating periods of slow diffusion (1.2-s lifetime) and actin-dependent temporary immobilization called “Stimulation-induced Temporary Arrest of LateraL diffusion” (STALL) (0.57-s lifetime) in areas of ~50 nm in diameter (20–40% of the entire trajectory length)
CHO-K1 cells and primary neurons cultured on coverslips of glass-base dishes were transfected with the appropriate cDNAs, and ACP-prion protein (PrP) and ACP-Thy1 expressed on the cell surface were covalently labeled with the ATTO594-conjugated acyl-CoA ligand

Summary

Introduction

Various membrane proteins and lipids in the plasma membrane (PM) have been found to undergo temporary entrapment or immobilization in meso-scale (a few nm ~ a few 100 nm) domains [1,2,3,4,5,6,7,8]. Non-raft molecules often exhibited temporary immobilization/entrapment in meso-scale domains and molecular clusters in the PM [3, 5, 9]. Sahl et al [3] found that fluorescently-labeled sphingomyelin and phosphatidylethanolamine (with a hydrophobic ATTO647N dye in the headgroup), which both turned out to behave like non-raft lipids according to Sezgin et al [10] including the same authors, exhibited alternating periods of rapid diffusion and transient cholesterol-dependent entrapment/anchorage in areas of ~6 nm in diameter, with average trapping times of ~15 and

Methods

Results

Conclusion