Cuiwen He

High-resolution imaging and quantification of plasma membrane cholesterol by NanoSIMS

Significance Biochemical studies have demonstrated that one pool of cholesterol in the plasma membrane is accessible to binding by bacterial cholesterol-binding proteins, whereas another pool is “sequestered” and inaccessible to binding by those proteins. Here, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, along with cholesterol-binding proteins that had been labeled with a stable isotope, to visualize and quantify the distribution of “accessible cholesterol” on the plasma membrane of mammalian cells. Our studies revealed that accessible cholesterol, as judged by cholesterol-binding proteins, is not evenly distributed on the plasma membrane but instead is enriched on the surface of microvilli. The accessible cholesterol on microvilli could be relevant to the movement of cholesterol away from the plasma membrane. Cholesterol is a crucial lipid within the plasma membrane of mammalian cells. Recent biochemical studies showed that one pool of cholesterol in the plasma membrane is “accessible” to binding by a modified version of the cytolysin perfringolysin O (PFO*), whereas another pool is sequestered by sphingomyelin and cannot be bound by PFO* unless the sphingomyelin is destroyed with sphingomyelinase (SMase). Thus far, it has been unclear whether PFO* and related cholesterol-binding proteins bind uniformly to the plasma membrane or bind preferentially to specific domains or morphologic features on the plasma membrane. Here, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, in combination with 15N-labeled cholesterol-binding proteins (PFO* and ALO-D4, a modified anthrolysin O), to generate high-resolution images of cholesterol distribution in the plasma membrane of Chinese hamster ovary (CHO) cells. The NanoSIMS images revealed preferential binding of PFO* and ALO-D4 to microvilli on the plasma membrane; lower amounts of binding were detectable in regions of the plasma membrane lacking microvilli. The binding of ALO-D4 to the plasma membrane was virtually eliminated when cholesterol stores were depleted with methyl-β-cyclodextrin. When cells were treated with SMase, the binding of ALO-D4 to cells increased, largely due to increased binding to microvilli. Remarkably, lysenin (a sphingomyelin-binding protein) also bound preferentially to microvilli. Thus, high-resolution images of lipid-binding proteins on CHO cells can be acquired with NanoSIMS imaging. These images demonstrate that accessible cholesterol, as judged by PFO* or ALO-D4 binding, is not evenly distributed over the entire plasma membrane but instead is highly enriched on microvilli.

Monoclonal antibodies that bind to the Ly6 domain of GPIHBP1 abolish the binding of LPL

GPIHBP1, an endothelial cell protein, binds LPL in the interstitial spaces and shuttles it to its site of action inside blood vessels. For years, studies of human GPIHBP1 have been hampered by an absence of useful antibodies. We reasoned that monoclonal antibodies (mAbs) against human GPIHBP1 would be useful for 1) defining the functional relevance of GPIHBP1’s Ly6 and acidic domains to the binding of LPL; 2) ascertaining whether human GPIHBP1 is expressed exclusively in capillary endothelial cells; and 3) testing whether GPIHBP1 is detectable in human plasma. Here, we report the development of a panel of human GPIHBP1-specific mAbs. Two mAbs against GPIHBP1’s Ly6 domain, RE3 and RG3, abolished LPL binding, whereas an antibody against the acidic domain, RF4, did not. Also, mAbs RE3 and RG3 bound with reduced affinity to a mutant GPIHBP1 containing an Ly6 domain mutation (W109S) that abolishes LPL binding. Immunohistochemistry studies with the GPIHBP1 mAbs revealed that human GPIHBP1 is expressed only in capillary endothelial cells. Finally, we created an ELISA that detects GPIHBP1 in human plasma. That ELISA should make it possible for clinical lipidologists to determine whether plasma GPIHBP1 levels are a useful biomarker of metabolic or vascular disease

NanoSIMS imaging: an approach for visualizing and quantifying lipids in cells and tissues

Over the past few decades, several approaches have been used to image lipids in cells and tissues, but most have limited spatial resolution and sensitivity. Here, we discuss a relatively new approach, nanoscale secondary ion mass spectrometry imaging, that makes it possible to visualize lipids in cells and tissues in a quantitative fashion and with high spatial resolution and high sensitivity.

NanoSIMS Analysis of Intravascular Lipolysis and Lipid Movement across Capillaries and into Cardiomyocytes

The processing of triglyceride-rich lipoproteins (TRLs) in capillaries provides lipids for vital tissues, but our understanding of TRL metabolism is limited, in part because TRL processing and lipid movement have never been visualized. To investigate the movement of TRL-derived lipids in the heart, mice were given an injection of [2H]triglyceride-enriched TRLs, and the movement of 2H-labeled lipids across capillaries and into cardiomyocytes was examined by NanoSIMS. TRL processing and lipid movement in tissues were extremely rapid. Within 30 s, TRL-derived lipids appeared in the subendothelial spaces and in the lipid droplets and mitochondria of cardiomyocytes. Enrichment of 2H in capillary endothelial cells was not greater than in cardiomyocytes, implying that endothelial cells may not be a control point for lipid movement into cardiomyocytes. Remarkably, a deficiency of the putative fatty acid transport protein CD36, which is expressed highly in capillary endothelial cells, did not impede entry of TRL-derived lipids into cardiomyocytes.

An LPL-specific monoclonal antibody, 88B8, that abolishes the binding of LPL to GPIHBP1.

LPL contains two principal domains: an amino-terminal catalytic domain (residues 1–297) and a carboxyl-terminal domain (residues 298–448) that is important for binding lipids and binding glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1) (an endothelial cell protein that shuttles LPL to the capillary lumen). The LPL sequences required for GPIHBP1 binding have not been examined in detail, but one study suggested that sequences near LPL’s carboxyl terminus (residues ∼403–438) were crucial. Here, we tested the ability of LPL-specific monoclonal antibodies (mAbs) to block the binding of LPL to GPIHBP1. One antibody, 88B8, abolished LPL binding to GPIHBP1. Consistent with those results, antibody 88B8 could not bind to GPIHBP1-bound LPL on cultured cells. Antibody 88B8 bound poorly to LPL proteins with amino acid substitutions that interfered with GPIHBP1 binding (e.g., C418Y, E421K). However, the sequences near LPL’s carboxyl terminus (residues ∼403–438) were not sufficient for 88B8 binding; upstream sequences (residues 298–400) were also required. Additional studies showed that these same sequences are required for LPL binding to GPIHBP1. In conclusion, we identified an LPL mAb that binds to LPL’s GPIHBP1-binding domain. The binding of both antibody 88B8 and GPIHBP1 to LPL depends on large segments of LPL’s carboxyl-terminal domain.

Macrophages release plasma membrane-derived particles rich in accessible cholesterol

Significance Earlier studies suggested that particles are released from the macrophage plasma membrane, but the mechanism has been unclear. We found that filopodia of macrophages release large numbers of vesicular particles. Nanoscale secondary ion mass spectrometry revealed that these particles are enriched in cholesterol, including the “accessible” pool of cholesterol detectable by the cholesterol-binding protein. The cholesterol content of macrophage particles increased when the cells were loaded with cholesterol and could be depleted by incubating the cells with high-density lipoproteins. Our studies suggest that the release of particles by macrophages could be one mechanism for cholesterol efflux and that particles could be an intermediate in the movement of cholesterol to high-density lipoproteins. Macrophages are generally assumed to unload surplus cholesterol through direct interactions between ABC transporters on the plasma membrane and HDLs, but they have also been reported to release cholesterol-containing particles. How macrophage-derived particles are formed and released has not been clear. To understand the genesis of macrophage-derived particles, we imaged mouse macrophages by EM and nanoscale secondary ion mass spectrometry (nanoSIMS). By scanning EM, we found that large numbers of 20- to 120-nm particles are released from the fingerlike projections (filopodia) of macrophages. These particles attach to the substrate, forming a “lawn” of particles surrounding macrophages. By nanoSIMS imaging we showed that these particles are enriched in the mobile and metabolically active accessible pool of cholesterol (detectable by ALO-D4, a modified version of a cholesterol-binding cytolysin). The cholesterol content of macrophage-derived particles was increased by loading the cells with cholesterol or by adding LXR and RXR agonists to the cell-culture medium. Incubating macrophages with HDL reduced the cholesterol content of macrophage-derived particles. We propose that release of accessible cholesterol-rich particles from the macrophage plasma membrane could assist in disposing of surplus cholesterol and increase the efficiency of cholesterol movement to HDL.

NanoSIMS imaging reveals unexpected heterogeneity in nutrient uptake by brown adipocytes

Heterogeneity in the metabolic properties of adipocytes in white adipose tissue has been well documented. We sought to investigate metabolic heterogeneity in adipocytes of brown adipose tissue (BAT), focusing on heterogeneity in nutrient uptake. To explore the possibility of metabolic heterogeneity in brown adipocytes, we used nanoscale secondary ion mass spectrometry (NanoSIMS) to quantify uptake of lipids in adipocytes interscapular BAT and perivascular adipose tissue (PVAT) after an intravenous injection of triglyceride-rich lipoproteins (TRLs) containing [2H]triglycerides (2H-TRLs). The uptake of deuterated lipids into brown adipocytes was quantified by NanoSIMS. We also examined 13C enrichment in brown adipocytes after administering [13C]glucose or 13C-labeled mixed fatty acids by gastric gavage. The uptake of 2H-TRLs-derived lipids into brown adipocytes was heterogeneous, with 2H enrichment in adjacent adipocytes varying by more than fourfold. We also observed substantial heterogeneity in 13C enrichment in adjacent brown adipocytes after administering [13C]glucose or [13C]fatty acids by gastric gavage. The uptake of nutrients by adjacent brown adipocytes within a single depot is variable, suggesting that there is heterogeneity in the metabolic properties of brown adipocytes.

Lipoprotein lipase reaches the capillary lumen in chickens despite an apparent absence of GPIHBP1

In mammals, GPIHBP1 is absolutely essential for transporting lipoprotein lipase (LPL) to the lumen of capillaries, where it hydrolyzes the triglycerides in triglyceride-rich lipoproteins. In all lower vertebrate species (e.g., birds, amphibians, reptiles, fish), a gene for LPL can be found easily, but a gene for GPIHBP1 has never been found. The obvious question is whether the LPL in lower vertebrates is able to reach the capillary lumen. Using purified antibodies against chicken LPL, we showed that LPL is present on capillary endothelial cells of chicken heart and adipose tissue, colocalizing with von Willebrand factor. When the antibodies against chicken LPL were injected intravenously into chickens, they bound to LPL on the luminal surface of capillaries in heart and adipose tissue. LPL was released rapidly from chicken hearts with an infusion of heparin, consistent with LPL being located inside blood vessels. Remarkably, chicken LPL bound in a specific fashion to mammalian GPIHBP1. However, we could not identify a gene for GPIHBP1 in the chicken genome, nor could we identify a transcript for GPIHBP1 in a large chicken RNA-seq data set. We conclude that LPL reaches the capillary lumen in chickens - as it does in mammals - despite an apparent absence of GPIHBP1.