Abstract
Tetraspanins, including CD53 and CD81, are four-transmembrane proteins that affect the membrane organization to regulate cellular processes including migration, proliferation, and signaling. However, it is unclear how the organizing function of tetraspanins is regulated at the molecular level. Here, we investigated whether recently proposed "open" and "closed" conformations of tetraspanins regulate the nanoscale organization of the plasma membrane of B cells. We generated conformational mutants of CD53 (F44E) and CD81 (4A, E219Q) that represent the "closed" and "open" conformation, respectively. Surface expression of these CD53 and CD81 mutants was comparable to that of WT protein. Localization of mutant tetraspanins into nanodomains was visualized by super-resolution direct stochastic optical reconstruction microscopy. Whereas the size of these nanodomains was unaffected by conformation, the clustered fraction of "closed" CD53 was higher and of "open" CD81 lower than respective WT protein. In addition, KO cells lacking CD53 showed an increased likelihood of clustering of its partner CD45. Interestingly, "closed" CD53 interacted more with CD45 than WT CD53. Absence of CD81 lowered the cluster size of its partner CD19 and "closed" CD81 interacted less with CD19 than WT CD81, but "open" CD81 did not affect CD19 interaction. However, none of the tetraspanin conformations made significant impact on the nanoscale organization of their partners CD19 or CD45. Taken together, conformational mutations of CD53 and CD81 differentially affect their nanoscale organization, but not the organization of their partner proteins. This study improves the molecular insight into cell surface nanoscale organization by tetraspanins.
