AFM detects the effects of acidic condition on the size and biomechanical properties of native/oxidized low-density lipoprotein.

Solution acidification exists under some physiological conditions (e.g. lysosomes in cells) and diseases (e.g. atherosclerosis, tumors, etc.). It is poorly understood whether and how acidification influences the size and biomechanical (stiffness and stickiness) properties of native Low-density lipoprotein (LDL) and its oxidized form (oxLDL) which plays a vital role in atherogenesis and tumorigenesis. Atomic force microscopy (AFM) evaluated that gradient acidification from pH 7.4 to pH 4.4 caused an expanding-first-and-then-shrinking decrease in size and a dramatic decrease in stiffness (but no statistically significant changes in stickiness) of LDL/oxLDL particles by influencing secondary/tertiary structures and lipid release detected by infrared spectral analysis and cholesterol detection, respectively. The smaller and softer characteristics of LDL/oxLDL at acidic conditions versus at the neutral pH partially explains the atherogenic role of acidification. The data may provide important information for a better understanding of LDL/oxLDL and some diseases (e.g. atherosclerosis and tumors).

Comparative investigation on the sizes and scavenger receptor binding of human native and modified lipoprotein particles with atomic force microscopy.

BACKGROUND: The size and receptor-binding abilities of plasma lipoproteins are closely related with their structure/functions. Presently, the sizes of native lipoproteins have been measured by various methods including atomic force microscopy (AFM) whereas the sizes of modified lipoproteins are poorly determined and the receptor-binding ability of lipoproteins is less detected and compared at the nanoscale. METHODS: Here, AFM was utilized to detect/compare the size and scavenger receptor-binding properties of three native human lipoproteins including high-density lipoprotein, low-density lipoprotein (LDL), and very low-density lipoprotein, and two modified human lipoproteins including oxidized and acetylated LDL, as well as bovine serum albumin and their antibodies as negative and positive controls, respectively. RESULTS: AFM detected that the sizes of these lipoproteins are close to the commonly known values and the previously-reported AFM-detected sizes and that native and modified LDL have different height/size. AFM also revealed that the CD36-binding abilities of the five lipoproteins are different from one another and from their SR-B1-binding abilities and that the anti-CD36/SR-B1 antibodies as positive controls have strong CD36/SR-B1-binding abilities. CONCLUSIONS: The data provide important information on lipoproteins for better understanding their structures/functions. Moreover, the data certify that besides size measurement AFM also can visualize receptor-lipoprotein binding at the nanoscale, as well as antigen-antibody (scavenger receptors and their antibodies) binding.

In situ AFM imaging of apolipoprotein A-I directly derived from plasma HDL.

BACKGROUND AND AIMS: The major apolipoproteins of plasma lipoproteins play vital roles in the structural integrity and physiological functions of lipoproteins. More than ten structural models of apolipoprotein A-I (apoA-I), the major apolipoprotein of high-density lipoprotein (HDL), have been developed successively. In these models, apoA-I was supposed to organize in a ring-shaped form. To date, however, there is no direct evidence under physiological condition. METHODS: Here, atomic force microscopy (AFM) was used to in situ visualize the organization of apoA-I, which was exposed via depletion of the lipid component of plasma HDL pre-immobilized on functionalized mica sheets. RESULTS: For the first time, the ring-shaped coarse structure and three detailed structures (crescent-shaped, gapped "O"-shaped, and parentheses-shaped structures, respectively) of apoA-I in plasma HDL, which have the ability of binding scavenger receptors, were directly observed and quantitatively measured by AFM. The three detailed structures probably represent the different extents to which the lipid component of HDL was depleted. Data on lipid depletion of HDL may provide clues to understand lipid insertion of HDL. CONCLUSIONS: These data provide important information for the understanding of the structure/maturation of plasma HDL. Moreover, they suggest a powerful method for directly visualizing the major apolipoproteins of plasma lipoproteins or the protein component of lipoprotein-like lipid-protein complexes.

Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation.

Cyclodextrins (CDs) have long been widely used as drug/food carriers and were recently developed as drugs for the treatment of diseases (e.g. Niemann-Pick C1 and cancers). It is unknown whether cyclodextrins may influence the structure of low-density lipoprotein (LDL), its susceptibility to oxidation, and atherogenesis. In this study, four widely used cyclodextrins including α-CD, γ-CD, and two derivatives of ß-CD (HPßCD and MßCD) were recruited. Interestingly, agarose gel electrophoresis (staining lipid and protein components of LDL with Sudan Black B and Coomassie brilliant blue, respectively but simultaneously) shows that cyclodextrins at relatively high concentrations caused disappearance of the LDL band and/or appearance of an additional protein-free lipid band, implying that cyclodextrins at relatively high concentrations can induce significant electrophoresis-detectable lipid depletion of LDL. Atomic force microscopy (AFM) detected that MßCD (as a representative of cyclodextrins) induced size decrease of LDL particles in a dose-dependent manner, further confirming the lipid depletion effects of cyclodextrins. Moreover, the data from agarose gel electrophoresis, conjugated diene formation, MDA production, and amino group blockage of copper-oxidized LDL show that cyclodextrins can impair LDL susceptibility to oxidation. It implies that cyclodextrins probably help to inhibit atherogenesis by lowering LDL oxidation.

Effects of membrane lipid composition and antibacterial drugs on the rigidity of Escherichia coli: Different contributions of various bacterial substructures.

The rigidity/stiffness is an important biomechanical property of bacteria and potentially correlated with many bacterial activities. While the rigidity or fluidity of the bacterial membrane has been extensively studied, the contributions of different bacterial substructures to the bacterial rigidity are less investigated. Here, we utilized four Escherichia coli (E. coli) strains with different membrane lipid compositions and three antibacterial drugs (EDTA, lysozyme, and streptomycin) to specifically alter bacterial substructures. By using atomic force microscopy (AFM), we found that the average height and Young's modulus of phosphatidylethanolamine (PE)-deficient E. coli strains were larger than those of PE(+) strains and that EDTA, EDTA plus lysozyme instead of lysozyme alone, and streptomycin all caused significant decreases in height and Young's modulus of the four E. coli strains. Our data imply that membrane lipid composition, the integrated outer membrane, the cell wall, and the cytoplasmic content are all responsible for bacterial rigidity but to different extents.

Imaging and force measurement of LDL and HDL by AFM in air and liquid.

The size and biomechanical properties of lipoproteins are tightly correlated with their structures/functions. While atomic force microscopy (AFM) has been used to image lipoproteins the force measurement of these nano-sized particles is missing. We detected that the sizes of LDL and HDL in liquid are close to the commonly known values. The Young's modulus of LDL or HDL is ∼0.4 GPa which is similar to that of some viral capsids or nanovesicles but greatly larger than that of various liposomes. The adhesive force of LDL or HDL is small (∼200 pN). The comparison of AFM detection in air and liquid was also performed which is currently lacking. Our data may provide useful information for better understanding and AFM detection of lipoproteins.

AFM of the ultrastructural and mechanical properties of lipid-raft-disrupted and/or cold-treated endothelial cells.

The nonionic detergent extraction at 4 °C and the cholesterol-depletion-induced lipid raft disruption are the two widely used experimental strategies for lipid raft research. However, the effects of raft disruption and/or cold treatment on the ultrastructural and mechanical properties of cells are still unclear. Here, we evaluated the effects of raft disruption and/or cold (4 °C) treatment on these properties of living human umbilical vein endothelial cells (HUVECs). At first, the cholesterol-depletion-induced raft disruption was visualized by confocal microscopy and atomic force microscopy (AFM) in combination with fluorescent quantum dots. Next, the cold-induced cell contraction and the formation of end-branched filopodia were observed by confocal microscopy and AFM. Then, the cell-surface ultrastructures were imaged by AFM, and the data showed that raft disruption and cold treatment induced opposite effects on cell-surface roughness (a significant decrease and a significant increase, respectively). Moreover, the cell-surface mechanical properties (stiffness and adhesion force) of raft-disrupted- and/or cold-treated HUVECs were measured by the force measurement function of AFM. We found that raft disruption and cold treatment induced parallel effects on cell stiffness (increase) or adhesion force (decrease) and that the combination of the two treatments caused dramatically strengthened effects. Finally, raft disruption was found to significantly impair cell migration as previously reported, whereas temporary cold treatment only caused a slight but nonsignificant decrease in cell migration performed at physiological temperature. Although the mechanisms for causing these results might be complicated and more in-depth studies will be needed, our data may provide important information for better understanding the effects of raft disruption or cold treatment on cells and the two strategies for lipid raft research.