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Single-Molecule Diffusion Reveals Similar Mobility for the Lck, H-Ras, and K-Ras Membrane Anchors

Piet H. M. Lommerse ^* , Karin Vastenhoud ^* , Niina J. Pirinen , Anthony I. Magee , Herman P. Spaink  and Thomas Schmidt ^*

^* Physics of Life Processes, Leiden Institute of Physics, and Department of Molecular Cell Biology, Institute of Biology, Leiden University, Leiden, The Netherlands; and Division of Biomedical Sciences, Imperial College London, London, United Kingdom

Correspondence: Address reprint requests to T. Schmidt, Physics of Life Processes, Leiden Institute of Physics, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands. Tel.: 31-71-527-5982; Fax.: 31-71-527-5819; E-mail: schmidt{at}physics.leidenuniv.nl.

Recent evidence on the occurrence of small (5–700 nm diameter) lipid microdomains in the exoplasmic leaflet of the plasma membrane has evoked interest in the possibility that similar domains may also be present in the cytoplasmic leaflet of the plasma membrane. However, current knowledge about these "lipid rafts", in live cells is limited. One way to obtain insight into the occurrence and the size of lipid rafts is the use of single-molecule microscopy, which allows one to study the diffusive motion of individual molecules with high positional and temporal accuracy. Using this technique, we compared the diffusion behavior of the Lck membrane anchor, which has a high affinity for lipid rafts, to the diffusion behavior of the K-Ras membrane anchor, which has negligible affinity for rafts and compared the results with those of the H-Ras membrane anchor. Surprisingly, we found only minor differences in the diffusion behavior of the various lipid anchors, indicating that putative cytoplasmic leaflet lipid rafts would have to be small (<137 nm diameter) and do not affect the mobility of membrane-anchored molecules much on timescales up to 60 ms.