title: Deregulation of focal adhesion formation and cytoskeletal tension due to loss of A-type lamins.
abstract: The nuclear lamina mechanically integrates the nucleus with the cytoskeleton and extracellular environment and regulates gene expression. These functions are exerted through direct and indirect interactions with the lamina's major constituent proteins, the A-type lamins, which are encoded by the LMNA gene. Using quantitative stable isotope labeling-based shotgun proteomics we have analyzed the proteome of human dermal fibroblasts in which we have depleted A-type lamins by means of a sustained siRNA-mediated LMNA knockdown. Gene ontology analysis revealed that the largest fraction of differentially produced proteins was involved in actin cytoskeleton organization, in particular proteins involved in focal adhesion dynamics, such as actin-related protein 2 and 3 (ACTR2/3), subunits of the ARP2/3 complex, and fascin actin-bundling protein 1 (FSCN1). Functional validation using quantitative immunofluorescence showed a significant reduction in the size of focal adhesion points in A-type lamin depleted cells, which correlated with a reduction in early cell adhesion capacity and an increased cell motility. At the same time, loss of A-type lamins led to more pronounced stress fibers and higher traction forces. This phenotype could not be mimicked or reversed by experimental modulation of the STAT3-IL6 pathway, but it was partly recapitulated by chemical inhibition of the ARP2/3 complex. Thus, our data suggest that the loss of A-type lamins perturbs the balance between focal adhesions and cytoskeletal tension. This imbalance may contribute to mechanosensing defects observed in certain laminopathies.
authors: a Laboratory of Cell Biology and Histology , Department of Veterinary Sciences, University of Antwerp , Antwerp , Belgium.|b Cell Systems and Imaging Research Group (CSI) , Department of Molecular Biotechnology, Ghent University , Ghent , Belgium.
authors: Tobias D J
authors: http://orcid.org/0000-0002-7604-4013
authors: TDJ
authors: Corne
authors: a Laboratory of Cell Biology and Histology , Department of Veterinary Sciences, University of Antwerp , Antwerp , Belgium.|b Cell Systems and Imaging Research Group (CSI) , Department of Molecular Biotechnology, Ghent University , Ghent , Belgium.
authors: Tom
authors: http://orcid.org/0000-0002-6529-2759
authors: T
authors: Sieprath
authors: c Medical Biotechnology Center, VIB , Belgium.|d Department of Biochemistry , Ghent University , Ghent , Belgium.
authors: Jonathan
authors: J
authors: Vandenbussche
authors: e Mechanobiology & Soft Matter Research Group, Interfaces and Complex Fluids Laboratory, Research Institute for Biosciences, University of Mons , Mons , Belgium.
authors: Danahe
authors: D
authors: Mohammed
authors: f Department of Cell Biology , Radboud University Medical Center , Nijmegen , The Netherlands.
authors: Mariska
authors: M
authors: Te Lindert
authors: c Medical Biotechnology Center, VIB , Belgium.|d Department of Biochemistry , Ghent University , Ghent , Belgium.
authors: Kris
authors: K
authors: Gevaert
authors: e Mechanobiology & Soft Matter Research Group, Interfaces and Complex Fluids Laboratory, Research Institute for Biosciences, University of Mons , Mons , Belgium.
authors: Sylvain
authors: http://orcid.org/0000-0001-8696-8052
authors: S
authors: Gabriele
authors: f Department of Cell Biology , Radboud University Medical Center , Nijmegen , The Netherlands.
authors: Katarina
authors: http://orcid.org/0000-0003-0616-2708
authors: K
authors: Wolf
authors: a Laboratory of Cell Biology and Histology , Department of Veterinary Sciences, University of Antwerp , Antwerp , Belgium.|b Cell Systems and Imaging Research Group (CSI) , Department of Molecular Biotechnology, Ghent University , Ghent , Belgium.
authors: Winnok H
authors: http://orcid.org/0000-0003-0960-6781
authors: WH
authors: De Vos
mesh_terms: D051377:Actin-Related Protein 2 / Q000235:genetics / Q000378:metabolism; D051376:Actin-Related Protein 2-3 Complex / Q000235:genetics / Q000378:metabolism; D051378:Actin-Related Protein 3 / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D005347:Fibroblasts; D022001:Focal Adhesions / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D034904:Lamin Type A / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D050796:STAT3 Transcription Factor / Q000235:genetics / Q000378:metabolism; D014945:Wound Healing / Q000502:physiology
chemical_list: D051377:Actin-Related Protein 2; D051376:Actin-Related Protein 2-3 Complex; D051378:Actin-Related Protein 3; C464459:CLIC4 protein, human; D002352:Carrier Proteins; D018118:Chloride Channels; C502658:FSCN1 protein, human; D015850:Interleukin-6; D034904:Lamin Type A; D008840:Microfilament Proteins; D020543:Proteome; D034741:RNA, Small Interfering; D050796:STAT3 Transcription Factor
keywords: LMNA; SILAC; cytoskeleton; focal adhesion; nuclear lamina; proteomics; stress fibers; wound healing