Cargando…

A method to map the interaction network of the nuclear lamina with genetically encoded photo-crosslinkers in vivo

Lamins are intermediate filaments that assemble in a meshwork at the inner nuclear periphery of metazoan cells. The nuclear periphery fulfils important functions by providing stability to the nuclear membrane, connecting the cytoskeleton with chromatin, and participating in signal transduction. Muta...

Descripción completa

Detalles Bibliográficos
Autores principales: Neumann-Staubitz, Petra, Kitsberg, Daniel, Buxboim, Amnon, Neumann, Heinz
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9468544/
https://www.ncbi.nlm.nih.gov/pubmed/36110135
http://dx.doi.org/10.3389/fchem.2022.905794
_version_ 1784788435945390080
author Neumann-Staubitz, Petra
Kitsberg, Daniel
Buxboim, Amnon
Neumann, Heinz
author_facet Neumann-Staubitz, Petra
Kitsberg, Daniel
Buxboim, Amnon
Neumann, Heinz
author_sort Neumann-Staubitz, Petra
collection PubMed
description Lamins are intermediate filaments that assemble in a meshwork at the inner nuclear periphery of metazoan cells. The nuclear periphery fulfils important functions by providing stability to the nuclear membrane, connecting the cytoskeleton with chromatin, and participating in signal transduction. Mutations in lamins interfere with these functions and cause severe, phenotypically diverse diseases collectively referred to as laminopathies. The molecular consequences of these mutations are largely unclear but likely include alterations in lamin-protein and lamin-chromatin interactions. These interactions are challenging to study biochemically mainly because the lamina is resistant to high salt and detergent concentrations and co-immunoprecipitation are susceptible to artefacts. Here, we used genetic code expansion to install photo-activated crosslinkers to capture direct lamin-protein interactions in vivo. Mapping the Ig-fold of laminC for interactions, we identified laminC-crosslink products with laminB1, LAP2, and TRIM28. We observed significant changes in the crosslink intensities between laminC mutants mimicking different phosphorylation states. Similarly, we found variations in laminC crosslink product intensities comparing asynchronous cells and cells synchronized in prophase. This method can be extended to other laminC domains or other lamins to reveal changes in their interactome as a result of mutations or cell cycle stages.
format Online
Article
Text
id pubmed-9468544
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-94685442022-09-14 A method to map the interaction network of the nuclear lamina with genetically encoded photo-crosslinkers in vivo Neumann-Staubitz, Petra Kitsberg, Daniel Buxboim, Amnon Neumann, Heinz Front Chem Chemistry Lamins are intermediate filaments that assemble in a meshwork at the inner nuclear periphery of metazoan cells. The nuclear periphery fulfils important functions by providing stability to the nuclear membrane, connecting the cytoskeleton with chromatin, and participating in signal transduction. Mutations in lamins interfere with these functions and cause severe, phenotypically diverse diseases collectively referred to as laminopathies. The molecular consequences of these mutations are largely unclear but likely include alterations in lamin-protein and lamin-chromatin interactions. These interactions are challenging to study biochemically mainly because the lamina is resistant to high salt and detergent concentrations and co-immunoprecipitation are susceptible to artefacts. Here, we used genetic code expansion to install photo-activated crosslinkers to capture direct lamin-protein interactions in vivo. Mapping the Ig-fold of laminC for interactions, we identified laminC-crosslink products with laminB1, LAP2, and TRIM28. We observed significant changes in the crosslink intensities between laminC mutants mimicking different phosphorylation states. Similarly, we found variations in laminC crosslink product intensities comparing asynchronous cells and cells synchronized in prophase. This method can be extended to other laminC domains or other lamins to reveal changes in their interactome as a result of mutations or cell cycle stages. Frontiers Media S.A. 2022-08-30 /pmc/articles/PMC9468544/ /pubmed/36110135 http://dx.doi.org/10.3389/fchem.2022.905794 Text en Copyright © 2022 Neumann-Staubitz, Kitsberg, Buxboim and Neumann. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Chemistry
Neumann-Staubitz, Petra
Kitsberg, Daniel
Buxboim, Amnon
Neumann, Heinz
A method to map the interaction network of the nuclear lamina with genetically encoded photo-crosslinkers in vivo
title A method to map the interaction network of the nuclear lamina with genetically encoded photo-crosslinkers in vivo
title_full A method to map the interaction network of the nuclear lamina with genetically encoded photo-crosslinkers in vivo
title_fullStr A method to map the interaction network of the nuclear lamina with genetically encoded photo-crosslinkers in vivo
title_full_unstemmed A method to map the interaction network of the nuclear lamina with genetically encoded photo-crosslinkers in vivo
title_short A method to map the interaction network of the nuclear lamina with genetically encoded photo-crosslinkers in vivo
title_sort method to map the interaction network of the nuclear lamina with genetically encoded photo-crosslinkers in vivo
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9468544/
https://www.ncbi.nlm.nih.gov/pubmed/36110135
http://dx.doi.org/10.3389/fchem.2022.905794
work_keys_str_mv AT neumannstaubitzpetra amethodtomaptheinteractionnetworkofthenuclearlaminawithgeneticallyencodedphotocrosslinkersinvivo
AT kitsbergdaniel amethodtomaptheinteractionnetworkofthenuclearlaminawithgeneticallyencodedphotocrosslinkersinvivo
AT buxboimamnon amethodtomaptheinteractionnetworkofthenuclearlaminawithgeneticallyencodedphotocrosslinkersinvivo
AT neumannheinz amethodtomaptheinteractionnetworkofthenuclearlaminawithgeneticallyencodedphotocrosslinkersinvivo
AT neumannstaubitzpetra methodtomaptheinteractionnetworkofthenuclearlaminawithgeneticallyencodedphotocrosslinkersinvivo
AT kitsbergdaniel methodtomaptheinteractionnetworkofthenuclearlaminawithgeneticallyencodedphotocrosslinkersinvivo
AT buxboimamnon methodtomaptheinteractionnetworkofthenuclearlaminawithgeneticallyencodedphotocrosslinkersinvivo
AT neumannheinz methodtomaptheinteractionnetworkofthenuclearlaminawithgeneticallyencodedphotocrosslinkersinvivo