**1. Introduction**

Tetraspanins are transmembrane proteins characterized by four membrane spanning hydrophobic domains, delineating two extracellular domains [1]. The large extracellular loop contains conserved residues, which contribute to the formation of a fold specific for tetraspanins [2]. Tetraspanins have been shown to modulate adhesion strengthening, cell migration, receptor signaling, cell–cell fusion [3] and trafficking of associated proteins [4]. Some tetraspanins, including CD82, have been associated with cancer progression [5–7]. This involvement in a wide spectrum of cellular processes can be explained by the ability of tetraspanins to form a network of protein–protein interactions within membranes, the socalled tetraspanin webs (or tetraspanin-enriched microdomains (TEM or TERM)) that rely on the homo- or hetero-multimerization of tetraspanins and stable interactions with other membrane proteins [8]. Importantly, using advanced fluorescence microscopy techniques, we and others have demonstrated that several tetraspanins (e.g., CD9, CD81 and CD151)

**Citation:** Fernandez, L.; Malrieu, M.; Bénistant, C.; Dosset, P.; Rubinstein, E.; Odintsova, E.; Berditchevski, F.; Milhiet, P.-E. CD82 and Gangliosides Tune CD81 Membrane Behavior. *Int. J. Mol. Sci.* **2021**, *22*, 8459. https:// doi.org/10.3390/ijms22168459

Academic Editor: Masoud Jelokhani-Niaraki

Received: 21 June 2021 Accepted: 3 August 2021 Published: 6 August 2021

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are dynamic molecules that can diffuse within the plasma membrane [9,10]. Using Single Molecule Tracking (SMT), two main modes of motion were identified: Brownian and confined with a combination of these two modes corresponding to the transient confinement of proteins [11,12]. Specifically, we demonstrated that tetraspanins are often confined within specific tetraspanins-enriched areas (TEAs) and that these areas form stable platforms in permanent exchange with the rest of the membrane [13]. Moreover, the functional relevance of this dynamics has been highlighted in the context of viral infection [14,15] and cell migration [16,17].

The tetraspanin CD82 (also known as KAI-1) has been identified as a metastasis suppressor in prostate cancer [18]. Subsequent studies demonstrated that the expression of CD82 protein is frequently lost or downregulated during tumor progression in various epithelial cancers [7]. While the role of CD82 in the modulation of cancer-/metastasisrelated pathways is not well understood, several studies have demonstrated that CD82 influences tumor cell migration and invasion by affecting integrin function or through association with EWI-2 and CD81 [19–22]. We also found that CD82 attenuates EGFinduced signaling [22] and regulates compartmentalization and dimerization of ErbB receptors [23]. Importantly, some of the CD82-dependent functions appear to be linked to gangliosides, glycosphingolipids found in the outer leaflet of the plasma membrane. Gangliosides have been proposed to form molecular assemblies containing tetraspanins and their partners (also referred to as glycosynapses [24]). In this regard, it has been demonstrated that various gangliosides modulate interactions of CD82 with its molecular partners [23]. Specifically, ganglioside depletion has a negative effect on the interaction between CD82 and EGFR, CD9, <sup>α</sup>3β1 integrin and CD151. In another study CD82 was shown to interact directly with the gangliosides GM2 and GM3, impairing cell migration by decreasing EGFR expression and phosphorylation and cMet activation [25]. Taken together, these observations support the idea that gangliosides could regulate, at least in part, the function of CD82-contaning complexes.

In this work, we examined how CD82 and gangliosides can affect the lateral organization of the tetraspanins CD9 and CD81 at the plasma membrane of non-malignant human mammary epithelial cell line (HB2 cells) using SMT, a technique pioneered by Schindler's group in biological membranes using dye molecules [26], allowing the observation of the motion of individual particles. This technique has been applied to investigate the motion of lipids and proteins within membranes and largely used in the context of raft microdomains (see the review [27]). As mentioned above, we already implemented in our group such a technique to study tetraspanin lateral segregation based on the use of monovalent Fab fragments directly labelled with a chemical dye to prevent the potential steric hindrance sometimes observed with quantum dots [28]. We used here SMT, combined with an automatic classification of membrane behavior of single transmembrane proteins based on neural network [29], to compare the membrane behavior of CD82 with that of two other tetraspanins, CD9 and CD81. Our experiments revealed that despite overlapping molecular partnerships, all three tetraspanin proteins display significant differences in their behavior at the plasma membrane. Specifically, we demonstrate that while CD82 expression increases the dynamics of CD81 and modifies its localization at the plasma membrane, the behavior and cellular distribution of CD9 is not modified. Finally, our results sugges<sup>t</sup> that gangliosides are not involved in CD82-dependent changes of CD81 dynamics but rather modulate the overall organization of tetraspanins into TEAs.
