A combined immuno-electron microscopy-statistical approach was used to directly visualize morphologically featureless plasma membrane microdomains. Plasma membrane sheets from HeLa cells were typically found at the margins of cells and characterized by their smooth homogeneous shading compared to the grainy background of the grid (). Immuno-gold labeling of validated raft (GFP-truncated H-ras; GFP-tH) and non-raft/disordered (GFP-truncated K-ras; GFP-tK) microdomain markers appeared indistinguishable (). However, statistical analysis of plasma membrane sheets revealed differences in the extent of clustering tendency and size of gold-labeled clusters ()
. GFP-tH is targeted by a combination of palmitoylation and a farnesylated CAAX motif to lipid rafts [6
]. K-function analysis of GFP-tH sheets revealed that the gold pattern was clustered, i.e., the curves exhibited a significant positive deviation from the L(r) - r = 0 value expected for a random point pattern (data not shown). Interestingly, both DHA and LA treatments enhanced (p<0.05) the curve peak heights, i.e. exhibited increased clustering of the lipid raft probe, as compared to OA and untreated cells (&). In contrast, analysis of GFP-tK labeled gold patterns revealed clustering, but with different characteristics relative to GFP-tH (&). Whilst superficially both LA and OA decreased total GFP-tK clustering (), pairwise analysis of GFP-tK K-functions revealed that only LA treatment significantly (p<0.05) reduced clustering in non-raft regions of plasma membranes (). Interestingly, the DHA-induced shift to the left of the curve for GFP-tK compared to untreated control indicates that non-raft cluster sizes also appear to be decreased in the presence of this lipid (). Collectively, these data suggest that the plasma membrane organization of inner leaflets is fundamentally altered by polyunsaturated fatty acids. Specifically, PUFA increase clustering of proteins in cholesterol-dependent microdomains (GFP-tH), whereas non-raft microdomains are insensitive to n-3-PUFA modulation.
Electron microscopy imaging of microdomains within 2D plasma membrane sheets
Analysis of inner-leaflet raft (tH) and non-raft (tK) markers
Difference between inner-leaflet raft (tH) treatments: Pairwise comparison plots
Difference between inner-leaflet non-raft (tK) treatments: Pairwise comparison plots
It is now appreciated that dietary PUFA are incorporated into both cholesterol/sphingolipid-rich detergent-resistant liquid ordered (lo
) and liquid disordered (ld
) plasma membrane microdomains in many cell types [11
]. For example, DHA is enriched 2−3 fold in both raft (0.6 to 5.3 mol%) and non-raft (2.4 to 7.6 mol%) domains following incorporation into the diet [11
]. The poor affinity of DHA and perhaps other long chain PUFA for cholesterol provides a lipid-driven mechanism for lateral phase separation of cholesterol/sphingolipid-rich lipid microdomains from the surrounding ld
phase in model membranes [7
]. This could alter the size, stability and distribution of cell surface lipid microdomains such as rafts. Indeed, it has been proposed that microdomain enrichment of PUFA may alter the dynamic partitioning of acylated proteins, thereby disrupting signal transduction events required for cell proliferation, apoptosis and differentiation [9
]. However, the ability of DHA and other fatty acids to influence lateral organization of lipid raft microdomains in situ
has not been determined to date. Experimental outcomes regarding biochemical isolation of microdomains vary depending on the isolation method, choice of detergent and cell type [5
]. To circumvent some of the problems associated with these we chose to utilize a more direct electron microscopic statistical approach in order to generate 2-D spatial maps with nanometer scale resolution of inner leaflet cell surface microdomains. In general, electron microscopy reveals a more complex and dynamic topographical organization of membrane microdomains than is predicted by biochemical analysis of detergent-resistant membranes [28
]. In a major step toward developing a unifying mechanistic hypothesis addressing how dietary PUFA modulate cell membrane microdomains, we demonstrate for the first time that DHA and LA differentially affect inner leaflet rafts and non-raft membrane microdomains. These findings highlight a novel modality by which PUFA influence membrane micro-organization.
In conclusion, we have shown for the first time that DHA and LA, major dietary fatty acids, differentially modulate inner leaflet cholesterol-dependent versus cholesterol-independent membrane microdomains. This is significant because the health benefits of select fatty acids are diverse and nutritional studies continue to demonstrate important benefits from the consumption of n-3 PUFA-enriched oils. Recently, the U.S. Food and Drug Administration (FDA) has approved the use of a health claim on labels for foods containing n-3 PUFA. Therefore, it is both appropriate and timely to precisely determine how DHA and other fatty acids modulate cell membrane structure/function.