Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It

Abstract

Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with cholesterol or hemagglutinin. The alternate views of plasma membrane organization suggested by these findings are discussed.

Characterization of Plasma Membrane Ceramides by Super‐Resolution Microscopy

“…Apart from the de novo synthesis,c eramides can also arise from the hydrolysis of sphingomyelin by the activation of sphingomyelinases (SMases). [1,2,[6][7][8][9] To study the influence of SMases on the distribution of ceramides in the plasma membrane,w ei ncubated HBMEC with exogenous Bacillus cereus sphingomyelinase (bSMase) before imaging ( Figure 2). First, we performed flow cytometry measurements of HBMEC incubated with different concentrations of bSMase before staining with anti-ceramide antibodies.H ere,t he highest ceramide concentration in the plasma membrane is visible with 150 mU bSMase during a40min incubation period (Supporting Information, Figure S7).…”

“…Furthermore,the fixation procedure can alter the distribution of molecules in the plasma membrane.E ven after fixation with 4% formaldehyde (FA) and 0.3 %glutaraldehyde (GA), residual post-fixation diffusion of membrane sphingolipids in combination with antibody crosslinking can potentially lead to clustering artifacts. [8,36,37] Therefore,infirst experiments we investigated the influence of different live and fixed cell labeling conditions on the distribution of plasma membrane ceramides in U2OS cells (Supporting Information, Figure S1)). In accordance with previous experiments, [33] our results demonstrate that independent of the labeling conditions,f or example,l ive-cell, or formaldehyde and glutaraldehyde fixation, respectively,c eramides form CRPs with similar distribution and size (Supporting Information, Figure S1).…”

Characterization of Plasma Membrane Ceramides by Super‐Resolution Microscopy

“…[3][4][5][6] Activation of SMases by cellular stress or ligation of receptors results in ceramide synthesis and subsequent self-association within the plasma membrane resulting in ceramide-rich platforms (CRPs). [7,8] The formation of CRPs rearranges the organization of the plasma membrane including clustering of diverse receptors and facilitates vesicle formation and fusion. [8,9] These changes induce differentiation, proliferation, growth arrest, and cell death.…”

“…[7,8] The formation of CRPs rearranges the organization of the plasma membrane including clustering of diverse receptors and facilitates vesicle formation and fusion. [8,9] These changes induce differentiation, proliferation, growth arrest, and cell death. [1,2] Moreover, SMases and ceramides have been shown to be critically involved in the internalization of pathogens.…”

“…[1,2,[6][7][8][9] To study the influence of SMases on the distribution of ceramides in the plasma membrane we incubated HBMEC with exogenous Bacillus cereus sphingomyelinase (bSMase) before imaging (Fig. 2).…”