A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly

Abstract

Condensed title:Reconstituting functional SUN proteins in artificial nuclear membranes. ABSTRACT (180/180 words)The linker of nucleoskeleton and cytoskeleton (LINC) is a conserved nuclear envelope-spanning molecular bridge that is responsible for the mechanical integration of the nucleus with the cytoskeleton. LINC complexes are formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Despite recent structural insights, our mechanistic understanding of LINC complex assembly remains limited by the lack of an experimental system for its in vitro reconstitution and manipulation.Here, we describe artificial nuclear membranes (ANMs) as a synthetic biology platform based on mammalian cell-free expression for the rapid reconstitution of SUN proteins in supported lipid bilayers. We demonstrate that SUN1 and SUN2 are oriented in ANMs with solvent-exposed C-terminal KASH-binding SUN domains. We also find that SUN2 possesses a single transmembrane domain, while SUN1 possesses three. Finally, SUN protein-containing ANMs bind synthetic KASH peptides, thereby reconstituting the LINC complex core. This work represents the first in vitro reconstitution of KASH-binding SUN proteins in supported lipid bilayers using cell-free expression, which will be invaluable for testing proposed models of LINC complex assembly and its regulation.Eukaryotic cells are defined by the presence of a genome-containing nucleus, the boundary of which is delineated by the nuclear envelope (NE), a specialized subdomain of the endoplasmic reticulum (ER) (Kite, 1913). The NE consists of concentric inner and outer nuclear membranes (INM and ONM, respectively) separated by a ~30-50 nm perinuclear space (PNS) that is contiguous with the ER lumen (Watson, 1959).While the ONM is an extension of the ER, a unique subset of proteins resides in the INM that interact with the nuclear lamina and chromatin within the nucleoplasm (Burke and Stewart, 2014).Fusion between the INM and ONM creates numerous aqueous channels throughout the NE that are occupied by nuclear pore complexes (NPCs), which are the primary sites of molecular exchange between the cytoplasm and nucleoplasm (Knockenhauer and Schwartz, 2016, Otsuka and Ellenberg, 2018). However, mechanical forces generated by the cytoskeleton within the cytoplasm can also be sensed and transmitted across the NE and into the nucleoplasm by LINC complexes (Alam et al., 2016, Brosig et al., 2010, Guilluy et al., 2014, Lombardi et al., 2011, Tajik et al., 2016. These evolutionarily conserved NE-spanning molecular bridges mediate several fundamental cellular processes including DNA damage repair, meiotic chromosome pairing, mechano-regulation of gene expression, and nuclear positioning (Alam et al., 2016, Chang et al., 2015, Meinke and Schirmer, 2015, Tapley and Starr, 2013. Consistent with their central role in cellular function is a growing list of genetic mutations in LINC complex proteins associated with human diseases such as aging-related hearing l...

SUN/KASH interactions facilitate force transmission across the nuclear envelope

“…For example, in vitro reconstituted LINC complexes on artificial nuclear membranes indicate that SUN2 contains a single transmembrane domain while SUN1 has three [41]. In addition, hydrophobic patches in the nucleoplasmic region of SUN proteins may associate with the inner nuclear membrane and the lamin network to distribute forces inside the nucleus [41]. Additional studies are required to determine how SUN and KASH domains might be directly interacting with the outer nuclear membrane.…”

“…Recent studies suggest possible mechanisms for LINC complexes to assemble into larger structures., Mammalian SUN1 proteins form oligomers larger than trimers detected in living cells by fluorescence fluctuation spectroscopy [49]. SUN1 has also been observed to form clusters in a reconstituted, cell-free expression system on artificial lipid bilayers [41]. Computer modeling predicts that SUN1 trimers can associate with each other laterally through their SUN domains to form higher-order clusters [41,43].…”

“…SUN1 has also been observed to form clusters in a reconstituted, cell-free expression system on artificial lipid bilayers [41]. Computer modeling predicts that SUN1 trimers can associate with each other laterally through their SUN domains to form higher-order clusters [41,43]. It has also been proposed that a cysteine adjacent to the SUN domain of SUN1 protein is able to form intermolecular disulfide bonds with neighboring SUN trimers [44,50].…”

In vitrosynthesis and reconstitution using mammalian cell-free lysates enables the systematic study of the regulation of LINC complex assembly

“…In our previous work, we developed a simple bottom-up synthetic biology platform based on a mammalian cell-free expression (CFE) system for the rapid reconstitution and study of LINC complex assembly using full-length SUN proteins (7). The most abundant isoforms of SUN proteins (SUN1 and SUN2) from mouse were expressed in the presence of supported lipid bilayers with excess membrane reservoirs (SUPER) templates (8) to mediate spontaneous reconstitution.…”

Engineering spatiotemporal organization and dynamics in synthetic cells

“…An emerging paradigm for membrane engineering relies on the synthesis and incorporation of membrane proteins in situ by the means of a cell‐free expression system. Both prokaryotic and mammalian cell‐free expression systems have been shown to successfully express structurally intact membrane proteins in mixed micelles or lipid bilayers (Shinoda et al, 2016; Majumder, Willey, et al 2019). Also, it has been demonstrated that mechanosensitive channel proteins can be successfully expressed and incorporated into synthetic cells (Garamella, Majumder, Liu, & Noireaux, 2019; Hindley et al, 2019; Majumder et al, 2017).…”