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Related Experiment Videos

Cell-matrix adhesions differentially regulate fascin phosphorylation.

J C Adams1, J D Clelland, G D Collett

  • 1Medical Research Council-Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom. dmcbjca@ucl.ac.uk

Molecular Biology of the Cell
|December 10, 1999
PubMed
Summary

Cell adhesion regulates fascin phosphorylation via protein kinase C (PKC), impacting cell structure. This discovery links extracellular matrix interactions to cytoskeletal organization through specific signaling pathways.

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Area of Science:

  • Cell Biology
  • Biochemistry
  • Molecular Biology

Background:

  • Cell adhesion to extracellular matrix (ECM) components influences cell behavior.
  • The actin-bundling protein fascin plays a key role in cytoskeletal dynamics.
  • Fascin's actin-binding activity is regulated by phosphorylation, affecting cell structure.

Purpose of the Study:

  • To investigate how cell adhesion to different ECM molecules (fibronectin, laminin-1, thrombospondin-1) affects fascin phosphorylation.
  • To determine the role of protein kinase C (PKC) in mediating these adhesion-dependent signaling events.
  • To elucidate the functional consequences of fascin phosphorylation on cell morphology and microspike formation.

Main Methods:

  • Utilized differentiated cell types (C2C12 myoblasts, LLC-PK1 epithelial cells).

Related Experiment Videos

  • Employed 12-tetradecanoyl phorbol 13-acetate (TPA) as a PKC activator and calphostin C/chelerythrine chloride as PKC inhibitors.
  • Analyzed fascin localization and phosphorylation using immunofluorescence and 2D gel electrophoresis.
  • Investigated mutant fascin proteins (GFP-fascin S39A, GFP-fascin S39D) to assess phosphorylation site function.
  • Used antibodies against integrin alpha5 subunit to probe signaling pathways.

    • Main Results:

    • Adhesion to fibronectin, but not laminin-1 or thrombospondin-1, induced fascin phosphorylation in a PKC-dependent manner.
    • TPA treatment also led to fascin phosphorylation and altered fascin distribution.
    • Non-phosphorylatable (S39A) and constitutively phosphorylated (S39D) fascin mutants showed distinct effects on cell spreading and microspike formation.
    • PKCalpha activity was linked to fibronectin-induced fascin phosphorylation and actin association.
    • Integrin alpha5 signaling was involved in fibronectin-mediated fascin-actin interactions.

    Conclusions:

    • Matrix-initiated, PKC-dependent phosphorylation of fascin at serine 39 is a novel mechanism coupling ECM adhesion to cytoskeletal organization.
    • This signaling pathway regulates the formation of fascin-dependent cellular structures like microspikes.
    • Differential binding to ECM components leads to distinct intracellular signaling outcomes affecting cell morphology.