Biochemical visualization of cell surface molecular clustering in living cells

Abstract

Many plasma membrane-resident molecules cluster with other molecules to collaborate in a variety of biological events. We herein report a sensitive and simple method to identify components of cell surface molecular clusters in living cells. This method includes a recently established reaction, called the enzymemediated activation of radical source (EMARS), to label molecules within a limited distance (Ϸ200 -300 nm) from the probed molecule on which HRP is set. Because the size of this active area is close to that of the reported membrane clusters, it is suggested that the labeled molecules cluster with the probed molecule in the same membrane domain. A combination of the EMARS reaction and antibody array analysis demonstrated that many kinds of receptor tyrosine kinases (RTKs) formed clusters with ␤1 integrin in HeLa S3 cells. A similar antibody array analysis after the EMARS reaction with three HRP-labeled antibodies against growth factor receptors showed the patterns of biotinylated RTKs to be substantially different from each other. These results suggest that different types of cell surface molecular clusters can thus be distinguished using the EMARS reaction. Therefore, the present ''biochemical visualization'' method is expected to be a powerful tool to elucidate molecular clustering on the cell surface of living cells in various contexts.ganglioside ͉ integrin ͉ microdomain ͉ radicals T he biological events through the plasma membranes, such as signal transmission, cell adhesion, and trafficking require the interactions between receptors, adhesion molecules, and signaling proteins. Recent studies have accumulated a line of evidence in which the functional components are distributed nonrandomly on the plasma membrane and exist as clusters in the nanometerscale domains (1). These membrane domains are formed by the clustering of particular membrane lipids and proteins and display a dynamic property of association and dissociation between interacting molecules that occurs continuously (2). It is therefore essential to identify what functional molecules cocluster in the native membrane, and how they collaborate to create their biological output.Among the many types of membrane domains proposed, the ''lipid rafts'' that are enriched in cholesterol, sphingolipid, GPI-anchored proteins, and the Src kinase family members have so far been most intensively investigated (3-5). It has been assumed that the lipid raft fractions are extracted from the rest of the plasma membrane based on the fact that the membrane domains are resistant to nonionic detergents, whereas the fluid membrane dissolves (6). However, the isolated materials are a mixture of heterogeneous microdomains that could include artificial products extracted during the process of homogenization with detergents. Therefore, it is impossible to identify what molecules cocluster in the same microdomain under the physiological conditions by means of the detergent-resistant membrane fractionation.Until now, four analytical strategies have been developed to analy...

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