Detergent-resistant membranes in HeLa cells. A comparative study with an electrochemical and lipidomic perspective

Abstract

Two procedures are compared for the isolation of detergent-resistant membranes (DRMs) from the HeLa model cell line. The isolation was based on application of Triton X-100 followed by 4 or 18 h ultracentrifugation in sucrose (5–42.5, % w) or Optiprep™ (10–25, % w) gradients. In the fractions obtained, the total amount of protein, cholesterol, and free thiols was evaluated using spectrophotometry. Increased protein as well as free thiol contents were demonstrated in higher density fractions. In contrast, the highest cholesterol levels were observed in light or medium heavy fractions with a low proportion of sucrose or Optiprep, especially after 18 h of centrifugation. For the sucrose gradient, we used voltammetric determination of the catalytic hydrogen evolution reaction at the Hg-electrode for individual fractions. The catalytic response, expressed as the height of the presodium wave, increased from light to heavy fractions corresponding to the protein content and/or other catalytically active species. The size of the DRMs or their associates ranged from 20 to 1000 nm, independently of the isolation protocol used. Proteins typically associated with DRMs such as caveolin and flotillin and characteristic for light and medium heavy gradient fractions, were determined using immunochemistry. We studied the subcellular localization of caveolin, flotillin, raftlin and transferrin, a control protein found intracellularly in the cytoplasm. Using confocal fluorescence microscopy, we confirmed the presence of caveolin and flotillin in the cytoplasmic membrane of HeLa cells. Raftlin was identified in both the membrane, and as part of the cell nucleus. We also performed untargeted lipidomic LC-MS analysis of the individual fractions of sucrose ultracentrifugation gradient obtained after 18 h. The predominant lipid subclasses were phosphatidylcholines and diacylglycerols. Apart from cholesterol and its ester, the rest of identified lipid classes was similar to that found in full HeLa cell lysates. The presented findings could be important for interpreting interlaboratory results and may be used as a guide for further studies on DRMs.