A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly
| Autor: | Sagardip Majumder, G. W. Gant Luxton, Allen P. Liu, Patrick T. Willey, Maxwell S. DeNies |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Nuclear Envelope LINC complex Biology Microtubules 03 medical and health sciences Synthetic biology medicine Humans Inner membrane Nuclear Matrix Nuclear membrane Cytoskeleton Lipid bilayer Chemistry Microfilament Proteins Intracellular Signaling Peptides and Proteins Membrane Proteins Nuclear Proteins Cell Biology Cell biology Transmembrane domain 030104 developmental biology medicine.anatomical_structure Membrane Synthetic Biology Microtubule-Associated Proteins Linker HeLa Cells |
| DOI: | 10.1101/415745 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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