RT Journal Article
SR Electronic
T1 A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 415745
DO 10.1101/415745
A1 Sagardip Majumder
A1 Patrick T. Willey
A1 Maxwell S. DeNies
A1 Allen P. Liu
A1 G.W. Gant Luxton
YR 2018
UL http://biorxiv.org/content/early/2018/09/13/415745.abstract
AB 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.ANMArtificial nuclear membraneCCCoiled-coilCFECell-free expressionCRDCentral rod domainDiI1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine PerchlorateDOGS-NTA-Ni1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)DOPC1,2-dioleoyl-sn-glycero-3-phosphatidylcholineDOPE1,2-dioleoyl-sn-glycero-3-phosphoethanolamineDOPS1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt)Egg-PAL-α-phosphatidic acid (Egg, Chicken) (sodium salt)EREndoplasmic reticulumFForwardFLFull-lengthKASHKlarsicht, ANC-1, SYNE homologyHRHydrophobic regionINMInner nuclear membraneKLDKinase, Ligase, DpnILDLuminal domainLINCLinker of nucleoskeleton and cytoskeletonNENuclear envelopeNDNucleoplasmic domainNPCNuclear pore complexONMOuter nuclear membranePNSPerinuclear spaceRReverseRERestriction enzymeRFURelative fluorescence unitsSUNSad1/UNC-84SUPERSupported bilayers with excess membrane reservoirSUVSmall unilamellar vesicleTMDTransmembrane domainTRITCTetramethylrhodamineWTWild-type