CD9, CD63, CD81, and CD82 are glycoproteins of unknown function which belong to the tetraspan superfamily. These molecules have short cytoplasmic sequences, four transmembrane domains and two unequal extracellular regions. Here, we show that these molecules are associated with each other on cell surface and with other glycoproteins such as very late antigen (VLA) integrins and HLA-DR antigens. Moreover, the VLA integrins and HLA-DR antigens were also found to be associated. The interactions of these molecules were analyzed by transfection experiments. It is demonstrated that overexpression of CD9 antigen in Raji cells leads to a lower efficiency of precipitation of CD81 and CD82, suggesting a direct interaction between these molecules. In these cells, the co-precipitation of CD81 and CD82 was not modified, suggesting that these tetraspans did not compete for association. However, in COS-7 cells, transfection of both CD81 and CD82 led to a marked reduction of the number of CD9/CD81 or CD9/CD82 complexes compared to single-transfected cells, and this was associated with the appearance of CD81/CD82 complexes. Therefore, in this cellular system, CD9 competes with CD81 and CD82 for association with the other tetraspan proteins. Finally, the tetraspans do not compete for the association with integrins or HLA-DR. Indeed, when CD9 was expressed in Raji cells, it was incorporated into the pre-existing complexes of these molecules with CD81 and CD82. These data suggest the existence of a tetraspan network which, by connecting several molecules, may organize the positioning of cell surface proteins and play a role in signal transduction, cell adhesion, and motility.
Engineered inorganic nanoparticles are essential components in the development of nanotechnologies. For applications in nanomedicine, particles need to be functionalized to ensure a good dispersibility in biological fluids. In many cases however, functionalization is not sufficient: the particles become either coated by a corona of serum proteins or precipitate out of the solvent. In the present paper, we show that by changing the coating of iron oxide nanoparticles from a low-molecular weight ligand (citrate ions) to small carboxylated polymers (poly(acrylic acid)), the colloidal stability of the dispersion is improved and the adsorption/internalization of iron toward living mammalian cells is profoundly affected. Citrate-coated particles are shown to destabilize in all fetal-calf-serum based physiological conditions tested, whereas the polymer coated particles exhibit an outstanding dispersibility as well as a structure devoid of protein corona. The interactions between nanoparticles and human lymphoblastoid cells are investigated by transmission electron microscopy and flow cytometry. Two types of nanoparticle/cell interactions are underlined. Iron oxides are found either adsorbed on the cellular membranes, or internalized into membrane-bound endocytosis compartments. For the precipitating citrate-coated particles, the kinetics of interactions reveal a massive and rapid adsorption of iron oxide on the cell surfaces. The quantification of the partition between adsorbed and internalized iron was performed from the cytometry data. The results highlight the importance of resilient adsorbed nanomaterials at the cytoplasmic membrane.
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