In situ imaging of detergent-resistant membranes by atomic force microscopy

Purified detergent-resistant membranes (DRMs) are powerful tools for the bi ochemical study of plasma membrane domains. To what extent these isolated D RMs correspond to native membrane domains remains, however, a matter of deb ate. The most immediate question to be answered concerns the in situ size r ange of DRMs, a determination that escapes classical microscopy techniques. Tn this study we show that in situ three dimensional images of a material as fragile as Triton X-100-treated cells can be obtained, in buffer, by tap ping mode atomic force microscopy. These images establish that, prior to th e isolation procedure, the detergent plasma membrane fragments form domains whose size frequently exceeds 15-20 mu m(2). This DRMs size range is about 1 order of magnitude higher than that estimated for the larger microdomain s of living cells, which strongly suggests that membrane microdomains rearr ange into larger DRMs during Triton X-100 treatment. Concomitantly, the ima ges also reveal the presence of the cytoskeleton, which is resistant to det ergent extraction, and suggest that, in situ, DRMs are associated with the membrane cytoskeleton. (C) 2000 Academic Press.