title: PKCδ localization at the membrane increases matrix traction force dependent on PLCγ1/EGFR signaling.
abstract: BACKGROUND
  During wound healing, fibroblasts initially migrate into the wound bed and later contract the matrix. Relevant mediators of transcellular contractility revealed by systems analyses are protein kinase c delta/myosin light chain-2 (PKCδ/MLC-2). PKCδ is activated by growth factor-driven PLCγ1 hydrolysis of phosphoinositide bisphosphate (PIP2) hydrolysis when it becomes tranlocated to the membrane. This leads to MLC-2 phosphorylation that regulates myosin for contractility. Furthermore, PKCδ n-terminus mediates PKCδ localization to the membrane in relative proximity to PLCγ1 activity. However, the role this localization and the relationship to its activation and signaling of force is not well understood. Therefore, we investigated whether the membrane localization of PKCδ mediates the transcellular contractility of fibroblasts.
  
  
  METHODS
  To determine PKCδ activation in targeted membrane locations in mouse fibroblast cells (NR6-WT), two PKCδ constructs were generated; PKCδ-CaaX with farnesylation moiety targeting PKCδ to the membrane and PKCδ-SaaX a non-targeting control.
  
  
  RESULTS
  Increased mean cell force was observed before and during EGF stimulation in fibroblasts expressing membrane-targeted PKCδ (PKCδ-CaaX) when analyzed with 2D cell traction force and 3D compaction of collagen matrix. This effect was reduced in cells deficient in EGFR/PLCy1 signaling. In cells expressing non-membrane targeted PKCδ (PKCδ-SaaX), the cell force exerted outside the ECM (extracellular matrix) was less, but cell motility/speed/persistence was increased after EGF stimulation. Change in cell motility and increased force exertion was also preceded by change in cell morphology. Organization of actin stress fibers was also decreased as a result of increasing membrane targeting of PKCδ.
  
  
  CONCLUSIONS
  From these results membrane tethering of PKCδ leads to increased force exertion on ECM. Furthermore, our data show PLCγ1 regulation of PKCδ, at least in part, drives transcellular contractility in fibroblasts.
authors.affiliation: Department of Pathology, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Joshua
authors.initials: J
authors.lastname: Jamison
authors.forename: Douglas
authors.initials: D
authors.lastname: Lauffenburger
authors.forename: James C-H
authors.initials: JC
authors.lastname: Wang
authors.forename: Alan
authors.initials: A
authors.lastname: Wells
mesh_terms: D000818:Animals; D001696:Biomechanical Phenomena / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D002465:Cell Movement / Q000187:drug effects; D003094:Collagen / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D005782:Gels; D006801:Humans; D051379:Mice; D051966:Phospholipase C gamma / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism
chemical_list: D005782:Gels; D003094:Collagen; D066246:ErbB Receptors; D051745:Protein Kinase C-delta; D051966:Phospholipase C gamma