*Article* **CD82 and Gangliosides Tune CD81 Membrane Behavior**

**Laurent Fernandez 1,†, Morgane Malrieu 1, Christine Bénistant 1, Patrice Dosset 1, Eric Rubinstein 2,3, Elena Odintsova 4, Fedor Berditchevski 4 and Pierre-Emmanuel Milhiet 1,5,\***


† Present address: ARNA (Nucleic Acids: Natural and Artificial Regulations), INSERM (National Institute of Health and Medical Research), CNRS (Centre National de la Recherche Scientifique), Université dé Bordeaux, UMR 5320, U1212, IECB, 33000 Bordeaux, France.

**Abstract:** Tetraspanins are a family of transmembrane proteins that form a network of protein– protein interactions within the plasma membrane. Within this network, tetraspanin are thought to control the lateral segregation of their partners at the plasma membrane through mechanisms involving specific lipids. Here, we used a single molecule tracking approach to study the membrane behavior of tetraspanins in mammary epithelial cells and demonstrate that despite a common overall behavior, each tetraspanin (CD9, CD81 and CD82) has a specific signature in terms of dynamics. Furthermore, we demonstrated that tetraspanin dynamics on the cell surface are dependent on gangliosides. More specifically, we found that CD82 expression increases the dynamics of CD81 and alters its localization at the plasma membrane, this has no effect on the behavior of CD9. Our results provide new information on the ability of CD82 and gangliosides to differentially modulate the dynamics and organization of tetraspanins at the plasma membrane and highlight that its lipid and protein composition is involved in the dynamical architecture of the tetraspanin web. We predict that CD82 may act as a regulator of the lateral segregation of specific tetraspanins at the plasma membrane while gangliosides could play a crucial role in establishing tetraspanin-enriched areas.

**Keywords:** tetraspanins; CD81; CD82; gangliosides; single-molecule tracking; microdomain; membrane diffusion; fluorescence microscopy
