title: PAK1 is involved in sensing the orientation of collagen stiffness gradients in mouse fibroblasts.
abstract: Migrating cells sense variations of stiffness in connective tissue matrices but how cells detect and respond to stiffness orientation is not defined. We examined cell extension formation on collagen with underlying support (vertical stiffness gradient) or on collagen laterally supported by nylon (lateral stiffness gradient). At 6 h after plating, cells plated on laterally-supported collagen exhibited >2-fold more abundant and ~2-fold longer cell extensions than cells plated on collagen with underlying support. We examined whether p21-activated kinase 1 (PAK1) influences extension formation that is dependent on the orientation of support. At 6 h after plating on collagen with underlying support, wild-type cell extensions were 40% shorter than PAK1 knockdown cells. In contrast, on laterally-supported collagen, wild-type cell extensions were 2-fold longer than PAK1 knockdown cells. In cells plated on laterally-supported collagen, there were ~2-fold reductions of collagen fiber alignment and compaction in PAK1 knockdown cells compared with wild-type cells. PAK1 knockdown did not affect collagen fiber alignment or compaction by cells plated on collagen with underlying support. Wild-type cells with lateral support of collagen exhibited 3-fold increases of phospho-myosin staining at 6h, which was 2-fold lower in PAK1 knockdown cells. In contrast, cells on collagen with underlying support showed no increase of phospho-myosin staining at any times. PAK1 knockdown did not affect α2 or β1 integrin expression or function. We conclude that PAK1 is involved in the ability of cells to sense the orientation of stiffness in collagen substrates and generate contractile forces that affect cell extension formation.
authors.affiliation: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada. Electronic address: v.pinto@utoronto.ca.
authors.forename: V I
authors.initials: VI
authors.lastname: Pinto
authors.affiliation: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada. Electronic address: hamid.mohammadi@utoronto.ca.
authors.forename: H
authors.initials: H
authors.lastname: Mohammadi
authors.affiliation: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
authors.forename: W S
authors.initials: WS
authors.lastname: Lee
authors.affiliation: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
authors.forename: A H
authors.initials: AH
authors.lastname: Cheung
authors.affiliation: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
authors.forename: C A
authors.initials: CA
authors.lastname: McCulloch
mesh_terms: D000818:Animals; D022081:Cell Surface Extensions / Q000235:genetics / Q000378:metabolism*; D003094:Collagen / Q000737:chemistry*; D055785:Gene Knockdown Techniques; D039421:Integrin alpha2 / Q000235:genetics / Q000378:metabolism*; D019012:Integrin beta1 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009218:Myosins / Q000235:genetics / Q000378:metabolism; D041681:NIH 3T3 Cells; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D039421:Integrin alpha2; D019012:Integrin beta1; D003094:Collagen; C515905:Pak1 protein, mouse; D054462:p21-Activated Kinases; D009218:Myosins
keywords: Cell extensions; Collagen remodeling; Constrained matrices; Physical boundaries; p21-Activated kinase