Direct Evidence of Lipid Rafts by in situ Atomic Force Microscopy

Abstract

Lipid rafts are membrane microdomains enriched with cholesterol, glycosphingolipids, and proteins. Although they are broadly presumed to play a pivotal role in various cellular functions, there are still fierce debates about the composition, functions, and even existence of lipid rafts. Here high-resolution and time-lapse in situ atomic force microscopy is used to directly confirm the existence of lipid rafts in native erythrocyte membranes. The results indicate some important aspects of lipid rafts: most of the lipid rafts are in the size range of 100-300 nm and have irregular shape; the detergent-resistant membranes consist of cholesterol microdomains and are not likely the same as the lipid rafts; cholesterol contributes significantly to the formation and stability of the protein domains; and Band III is an important protein of lipid rafts in the inner leaflet of erythrocyte membranes, indicating that lipid rafts are exactly the functional domains in plasma membrane. This work provides direct evidence of the presence, size, and main constitutive protein of lipid rafts at a resolution of a few nanometers, which will pave the way for studying their structure and functions in detail.

Nanoscale Quantifying the Effects of Targeted Drug on Chemotherapy in Lymphoma Treatment Using Atomic Force Microscopy

“…How to eliminate the influence of cellular curvature is the issue that needs to be solved in the studies of AFM-based cellular surface roughness [21]. For example, we may extract the cell membranes and attach them onto flat substrate (such as mica) [56], and then we may exactly obtain the roughness of cell membranes. Cellular physiological activities are dynamic essentially.…”

Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation

“…Surprisingly, very few papers have described cholesterolenriched domains. Among these, submicrometric domains were previously inferred by another group on unfixed ghosts by high-resolution atomic force microscopy upon cholesterol extraction by mbCD (100-300 nm in diameter [49]). Alternatively, cholesterol submicrometric domains have been directly visualized on HeLa cells by super-resolution microscopy (PALM) using Dronpa-theta-D4 (*240 nm in diameter) at 20°C [27], but not in fibroblasts by high-resolution ion mass spectrometry based on 18 Ocholesterol at 37°C [28].…”

AFM analysis of the multiple types of molecular interactions involved in rituximab lymphoma therapy on patient tumor cells and NK cells

“…Using the rituximab-conjugated tips, we could then simultaneously probe the CD20-rituximab and FcR-rituximab interactions. In fact, with the same protocol of tip functionalization, the tips can be used to measure the antibody-antigen interactions [19,25,26], which validated the activity of the Fab region of the antibodies linked to the AFM tip. However, the activity of the Fc region of the antibodies linked to the tip has not been largely reported.…”

“…The negative control and blocking experiments validated the specific FcR-rituximab interactions measured on NK cells using rituximab-conjugated tips. A range of antibodies have been linked to AFM tips to measure the antigen-antibody interaction forces such as antihuman serum albumin antibody [39], anti-histone H3 antibodies [25], anti-Band III antibodies [26], Herceptin [40], and anti-UCP1 antibodies [41]. In these studies, the interactions between the Fab region of antibodies linked to the AFM tip and the antigen were probed.…”