Cell-Free Expression Systems: Probing Nuclear Mechanotransduction Using Novel Engineering Platforms

Abstract

Cell-free expression (CFE) of proteins has become a versatile tool for in vitro reconstitution studies. Among the many advantages of using such systems, functional reconstitution of membrane proteins is a major improvement over traditional purification alternatives. In this work, we use a CFE system derived from HeLa cells to functionally reconstitute membrane proteins in artificial lipid bilayer membranes. SUN proteins are inner nuclear membrane proteins which have been shown to play a major role in nuclear mechanotransduction. SUN proteins bind to Nesprins that are known to interact with cytoskeletal filaments, via SUN-KASH (C-terminal peptide in Nesprins) binding which results in force transmission from the cytoskeleton to the nuclear lamina. While the assembly of SUN-KASH complex is known, the topology of SUN proteins in the inner nuclear membrane and their range of interactions with other nuclear membrane proteins remain unexplored due to several challenges with traditional cell biology approaches. Here, we expressed SUN proteins using our CFE system on lipid bilayer-coated beads in vitro as a platform for studying topology of membrane protein reconstitution. We present compelling evidence to confirm ER microsomes-mediated membrane protein reconstitution and show the versatility of our approach to probe the interaction of SUN proteins and their mutants with the membrane and investigate their orientation of insertion in the bilayer. Further we demonstrate functionality of reconstituted SUN proteins by investigating their binding to KASH peptides and other SUN mutants. The results presented here indicate the possibility of using CFE systems as tools for probing protein functions in vitro.