Tetraspanin Enriched Microdomains - Web Formation and Function
Final Report Abstract
Tetraspanins are a family of small, multi-span membrane proteins. They play multiple physiological and pathophysiological roles. The basis of multi-functionality likely is the tetraspanin web, an interaction network of tetraspanins and their primary interaction partners. Tetraspanins also form tetraspanin-enriched microdomains (TEMs) that are considered to be signaling platforms. However, it is unclear how tetraspanins exert their many functions, and how the web and TEMs are structured. Classically, tetraspanins are studied by biochemical approaches, but more recently also by superresolution microscopy, that finds tetraspanins concentrated in ⁓ 100 nm large clusters that apparently scatter randomly across the cell membrane. We aimed for describing TEMs by microscopy and for identifying factors that organize TEMs, and asked what interactions underlie the formation of the microscopic tetraspanin-clusters. We identify EWI-2, a primary interaction partner of the ubiquitously expressed tetraspanins CD81 and CD9, as a factor organizing CD81 assemblies into arranged crowds that can be considered as areas of enriched CD81 clusters. Moreover, we find parallels between the composition of biochemically and microscopically characterized tetraspanin assemblies. Finally, using CD81-dimerization as example, we show that increasing the affinity between interacting CD81s increases the fraction of CD81 molecules that have a neighbored CD81 molecule at a distance below 25 nm. On the other hand, weakening the interaction has no effect, and also cluster characteristics, as size, apparently are not affected by the propensity to form oligomers. Hence, tetraspanin oligomerization has some effect on clustering but is not the main driving force. In summary, microscopic clusters are equal to biochemical tetraspanin-assemblies, defining in their entirety the tetraspanin web. Cluster-agglomeration by EWI-2 enriches tetraspanins, which makes them a microscopic complement of TEMs. This provides a conceptual framework for describing TEMs by microscopic parameters. The microscopic classification of tetraspanin assemblies opens the possibility to correlate biochemistry and microscopy, which is important for advancing our picture of this enigmatic protein family, whose members play roles in a plethora of cellular functions, diseases, and pathogen infections. In the future, this will help using these molecules as targets for therapy in cancer and viral infections, where tetraspanins play important roles.
