Dataset Information

PKC? localization at the membrane increases matrix traction force dependent on PLC?1/EGFR signaling.

ABSTRACT:

Introduction

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 (PKC?/MLC-2). PKC? is activated by growth factor-driven PLC?1 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, PKC? n-terminus mediates PKC? localization to the membrane in relative proximity to PLC?1 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 PKC? mediates the transcellular contractility of fibroblasts.

Methods

To determine PKC? activation in targeted membrane locations in mouse fibroblast cells (NR6-WT), two PKC? constructs were generated; PKC?-CaaX with farnesylation moiety targeting PKC? to the membrane and PKC?-SaaX a non-targeting control.

Results

Increased mean cell force was observed before and during EGF stimulation in fibroblasts expressing membrane-targeted PKC? (PKC?-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 PKC? (PKC?-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 PKC?.

Conclusion

From these results membrane tethering of PKC? leads to increased force exertion on ECM. Furthermore, our data show PLC?1 regulation of PKC?, at least in part, drives transcellular contractility in fibroblasts.

SUBMITTER: Jamison J

PROVIDER: S-EPMC3796482 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

PKCδ localization at the membrane increases matrix traction force dependent on PLCγ1/EGFR signaling.

Jamison Joshua J Lauffenburger Douglas D Wang James C-H JC Wells Alan A

PloS one 20131014 10

<h4>Introduction</h4>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. Furt ...[more]

PMID: 24155954

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