title: High content image analysis of focal adhesion-dependent mechanosensitive stem cell differentiation.
abstract: Human mesenchymal stem cells (hMSCs) receive differentiation cues from a number of stimuli, including extracellular matrix (ECM) stiffness. The pathways used to sense stiffness and other physical cues are just now being understood and include proteins within focal adhesions. To rapidly advance the pace of discovery for novel mechanosensitive proteins, we employed a combination of in silico and high throughput in vitro methods to analyze 47 different focal adhesion proteins for cryptic kinase binding sites. High content imaging of hMSCs treated with small interfering RNAs for the top 6 candidate proteins showed novel effects on both osteogenic and myogenic differentiation; Vinculin and SORBS1 were necessary for stiffness-mediated myogenic and osteogenic differentiation, respectively. Both of these proteins bound to MAPK1 (also known as ERK2), suggesting that it plays a context-specific role in mechanosensing for each lineage; validation for these sites was performed. This high throughput system, while specifically built to analyze stiffness-mediated stem cell differentiation, can be expanded to other physical cues to more broadly assess mechanical signaling and increase the pace of sensor discovery.
authors.affiliation: Department of Bioengineering, University of California, 9500 Gilman Drive, MC 0695, La Jolla, San Diego, CA, USA. aengler@ucsd.edu.
authors.forename: Andrew W
authors.identifier: 0000-0002-7206-0964
authors.initials: AW
authors.lastname: Holle
authors.affiliation: Department of Bioengineering, University of California, 9500 Gilman Drive, MC 0695, La Jolla, San Diego, CA, USA. aengler@ucsd.edu.
authors.forename: Alistair J
authors.initials: AJ
authors.lastname: McIntyre
authors.affiliation: Department of Bioengineering, University of California, 9500 Gilman Drive, MC 0695, La Jolla, San Diego, CA, USA. aengler@ucsd.edu.
authors.forename: Jared
authors.initials: J
authors.lastname: Kehe
authors.affiliation: Department of Bioengineering, University of California, 9500 Gilman Drive, MC 0695, La Jolla, San Diego, CA, USA. aengler@ucsd.edu.
authors.forename: Piyumi
authors.initials: P
authors.lastname: Wijesekara
authors.affiliation: Department of Bioengineering, University of California, 9500 Gilman Drive, MC 0695, La Jolla, San Diego, CA, USA. aengler@ucsd.edu.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Young
authors.affiliation: Department of Bioengineering, University of California, 9500 Gilman Drive, MC 0695, La Jolla, San Diego, CA, USA. aengler@ucsd.edu.
authors.forename: Ludovic G
authors.initials: LG
authors.lastname: Vincent
authors.affiliation: Department of Bioengineering, University of California, 9500 Gilman Drive, MC 0695, La Jolla, San Diego, CA, USA. aengler@ucsd.edu and Sanford Consortium for Regenerative Medicine, La Jolla, CA 92093, USA.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Engler
mesh_terms: D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D022001:Focal Adhesions / Q000502:physiology*; D006801:Humans; D007089:Image Enhancement / Q000379:methods; D020935:MAP Kinase Signaling System / Q000502:physiology; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000502:physiology*; D057054:Molecular Imaging / Q000379:methods; D020543:Proteome / Q000378:metabolism*; D013314:Stress, Mechanical
chemical_list: D020543:Proteome