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

Sagardip Majumder, Patrick T. Willey, Maxwell S. DeNies, Allen P. Liu, G.W. Gant Luxton

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Article numberjcs219451
JournalJournal of cell science
Volume132
Issue number4
DOIs
StatePublished - Feb 1 2019

Fingerprint

Synthetic Biology
Nuclear Matrix
Cytoskeleton
Nuclear Envelope
Dissection
Artificial Membranes
Lipid Bilayers
Proteins
Carrier Proteins
Peptides

Keywords

  • Cell-free expression
  • Cytoskeleton complex
  • KASH
  • Linker of nucleoskeleton
  • Nuclear membrane
  • Reconstitution
  • SUN

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

Cite this

A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly. / Majumder, Sagardip; Willey, Patrick T.; DeNies, Maxwell S.; Liu, Allen P.; Luxton, G.W. Gant.

In: Journal of cell science, Vol. 132, No. 4, jcs219451, 01.02.2019.

Research output: Contribution to journalArticle

Majumder, Sagardip ; Willey, Patrick T. ; DeNies, Maxwell S. ; Liu, Allen P. ; Luxton, G.W. Gant. / A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly. In: Journal of cell science. 2019 ; Vol. 132, No. 4.
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