Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton

Abstract

Although much evidence suggests that the plasma membrane of eukaryotic cells is not homogenous, the precise architecture of this important structure has not been clear. Here we use transmission electron microscopy of plasma membrane sheets and specific probes to show that most or all plasma membrane-associated proteins are clustered in cholesterol-enriched domains ("islands") that are separated by "protein-free" and cholesterol-low membrane. These islands are further divided into subregions, as shown by the localization of "raft" and "non-raft" markers to specific areas. Abundant actin staining and inhibitor studies show that these structures are connected to the cytoskeleton and at least partially depend on it for their formation and/or maintenance.

Fig. 1.

Localization of membrane-associated proteins in T cells. (A) Membrane sheets from resting T cells (PLL surface after PP2 treatment) were biotinylated on SH groups. Biotinylated proteins were detected by using streptavidin-conjugated 5-nm gold particles. (Left) An unmodified image of a typical membrane sheet. (Center) The same image in pseudocolor with the gold particles shown in filled black circles. (Right) Graph showing clustering by Hopkins analysis. (B) Membrane sheets from activated T cells (I-E^k/MCC + B7.1 surface) were labeled with 5-nm gold particles for tyrosine phosphorylation (Left) and ubiquitinylation (Right). Images are shown in pseudocolor with the gold particles shown as filled black circles. Hopkins analyses for tyrosine phosphorylation (Left) and ubiquitinylation (Right) show significant clustering. The relationship between the EM stain and pseudocolor is shown in the false-color bar.

Fig. 2.

Localization of raft and non-raft markers. (A) Membrane sheets from resting (Left) and activated (Right) T cells expressing tagged non-raft and raft markers, which are shown in filled red circles (5-nm gold particles) and filled black circles (10-nm gold particles), respectively. Hopkins analyses show clustering for the non-raft marker (top graphs) and raft marker (middle graphs). The Ripley's K analyses (bottom graphs) show repulsion or explicit separation. (B) Membrane sheets from activated T cells infected with either a non-raft (Left) or raft (Right) marker were stained for the expressed marker with specific antibodies (10-nm gold particles, filled black circles) and for cholesterol with perfringolysin O labeled with 5-nm gold particles (filled red circles). Hopkins analyses for cholesterol show slight clustering (top graphs), whereas the raft and non-raft markers are strongly clustered (middle graphs). Ripley's K analyses show colocalization for raft and non-raft markers with cholesterol (bottom graphs). The relationship between EM stain and pseudocolor is shown in the false-color bar.

Fig. 3.

Cytoskeletal association of protein islands. (A) Membrane sheets from resting T cells were stained for actin (all six isoforms) with 5-nm gold particles (filled black circles). Hopkins analysis shows clustering (graph). (B) T cells were surface-biotinylated on NH₂ groups and cultured for 90 min with (Upper) or without (Lower) 20 μM latrunculin A. T cells were adhered to streptavidin-coated EM grids for an additional 60 min in the continued absence or presence of the drug and ripped by using a streptavidin-coated coverslip. In both cases, Hopkins analysis shows clustering for raft and non-raft markers in the untreated and latrunculin A-treated cells (top and middle graphs, respectively). Because of the high density of protein islands in the membrane sheets from untreated T cells, no explicit separation can be detected with Ripley's K function (bottom graphs). However, the markers still show separation in the latrunculin A-treated cells (bottom graphs). An obvious reduction in the number of protein islands after latrunculin A treatment is visible. The relationship between EM stain and pseudocolor is shown in the false-color bar.

Fig. 4.

The protein island model. In this model all membrane-associated proteins are clustered in protein islands (green lipids) that are surrounded by a sea of protein-free membrane (yellow lipids). The islands can be subdivided into raft and non-raft islands, which is also illustrated by their lipid composition (bright-green and dark-green lipids, respectively) and protein contents (gray and red proteins, respectively). Molecules move with high diffusion rates within the islands, and the islands themselves can move with significant restrictions in the membrane. The protein islands are connected to the cytoskeleton (orange), most likely by actin because it plays a crucial role in island formation and/or maintenance. We propose that theses islands can exchange proteins and lipids by hop diffusion when in physical contact.