In Situ Imaging of Detergent-Resistant Membranes by Atomic Force Microscopy Marie-Ce ´cile Giocondi,* Ve ´ronique Vie ´,* Eric Lesniewska,† Jean-Pierre Goudonnet,† and Christian Le Grimellec* ,1 *Centre de Biochimie Structurale, INSERM U414, 29 rue de Navacelles, 34090 Montpellier Cedex; and †Laboratoire de Physique, CNRS-URA 5027, UFR Sciences et Techniques, 9 rue Alain Savary, BP 400, 21011 Dijon Cedex, France Received January 13, 2000, and in revised form March 16, 2000 Purified detergent-resistant membranes (DRMs) are powerful tools for the biochemical study of plasma membrane domains. To what extent these isolated DRMs correspond to native membrane do- mains remains, however, a matter of debate. The most immediate question to be answered concerns the in situ size range of DRMs, a determination that escapes classical microscopy techniques. In this study we show that in situ three-dimensional im- ages of a material as fragile as Triton X-100-treated cells can be obtained, in buffer, by tapping mode atomic force microscopy. These images establish that, prior to the isolation procedure, the detergent plasma membrane fragments form domains whose size frequently exceeds 15–20 m 2 . This DRMs size range is about 1 order of magnitude higher than that estimated for the larger microdomains of living cells, which strongly suggests that membrane mi- crodomains rearrange into larger DRMs during Tri- ton X-100 treatment. Concomitantly, the images also reveal the presence of the cytoskeleton, which is resistant to detergent extraction, and suggest that, in situ, DRMs are associated with the membrane cytoskeleton. © 2000 Academic Press Key Words: surface topography; membrane do- mains; detergent; cytoskeleton; atomic force mi- croscopy; CV-1 cells. INTRODUCTION The organization of membranes in microdomains postulated 25 years ago (Morrisett et al., 1975) is now believed to play a key role in the expression and regulation of membrane functions (reviewed in Gla- ser, 1993; Jacobson et al., 1995; Simons and Ikonen, 1997; Brown and London, 1998). Fluorescence ex- periments, single particle tracking, or optical twee- zers experiments on living cells suggest that the size of microdomains can vary from 50 up to 1200 nm in diameter (Yechiel and Edidin, 1987; Tocanne et al., 1989; Kusumi and Sako, 1996; Simson et al., 1998; Varma and Mayor, 1998). Detergent-resistant membranes (DRMs) (Brown and London, 1998), also called detergent-insoluble glycolipid-enriched mem- branes (DIGs) (Simons and Ikonen, 1997), or TIMs for Triton-insoluble membranes (Liu et al., 1997), have been isolated from cell lysates of almost all mammalian cell types as mixtures of vesicles and membrane sheets. They contain a specific group of membrane proteins, glycosylphosphatidylinositol- anchored, involved in signal transduction and are enriched in sphingolipids and cholesterol which form liquid-ordered phase (Lo) domains at the origin of the detergent insolubility (Brown and London, 1997; Harder and Simons, 1997; Brown, 1998). To what extent the isolated DRMs correspond to native membrane domains remains, however, a matter of debate (Simons and Ikonen, 1997; Brown, 1998; Hooper, 1999). A simple question still not answered concerns the size of DRMs in situ which might, or not, be compatible with that of microdomains. Atomic force microscopy (AFM) (Binnig et al., 1986) allows us to image the surface topography of biological samples in liquid with a resolution well beyond that of an optical microscope (Hoh and Han- sma, 1992), i.e., from molecular resolution on 2D crystals (Shao et al., 1996; Engel et al., 1997; Mu ¨ ller et al., 1998; Czajkowsky and Shao, 1998) to the imaging of the surface of a living cell with a lateral resolution better than 10 nm (Butt et al., 1990; Ho ¨rber et al., 1992; Le Grimellec et al., 1994, 1998). It therefore appears a good candidate for studying plasma membrane domains (Vie ´ et al., 1998). A lim- itation to AFM imaging, however, arises from the requirement for structures to develop sufficiently 1 To whom correspondence should be addressed at 29, rue de Navacelles, 34090 Montpellier Cedex, France. Fax: (33) 467 41 79 13. E-mail: clg@cbs.univ-montpl.fr. Journal of Structural Biology 131, 38 – 43 (2000) doi:10.1006/jsbi.2000.4266, available online at http://www.idealibrary.com on 38 1047-8477/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.