Journal Article

Spreading by snail (Lymnaea stagnalis) defence cells is regulated through integrated PKC, FAK and Src signalling

Walker, Anthony J, Mackintosh, David, Sealey, Katie L, Lacchini, Audrey H, Davies, Angela J

2010

Overview

Cell adhesion and spreading are vital to immune function. In molluscs, haemocytes (circulating phagocytes) are sentinels and effectors of the internal defence system; however, molecular mechanisms that regulate integrin-mediated spreading by haemocytes have not been characterised in detail. Visualisation of Lymnaea stagnalis haemocytes by scanning electron microscopy revealed membrane ruffling, formation of lamellipodia and extensive filopodia during early stages of cell adhesion and spreading. These events correlated with increased phosphorylation (activation) of protein kinase C (PKC) and focal adhesion kinase (FAK), sustained for 60 min. Treatment of haemocytes with the PKC inhibitors GF109203X or Gö 6976, or the Src/tyrosine kinase inhibitors SrcI or herbimycin A, attenuated haemocyte spread by 64, 46, 32 and 35%, respectively (P ≤ 0.001); PKC or Src inhibition also prevented focal adhesion formation. Western blotting demonstrated that during spreading and adhesion these inhibitors also impaired PKC and FAK activation, with Gö 6976 or SrcI inhibiting FAK phosphorylation by at least 70% (P ≤ 0.001), and herbimycin A or SrcI inhibiting PKC phosphorylation by at least 46% (P ≤ 0.01). Confocal microscopy revealed phosphorylated PKC colocalised with focal adhesion sites, particularly during early phases of adhesion and spreading. Finally, fibronectin promoted PKC and FAK phosphorylation in suspended haemocytes demonstrating that activation can occur independent of cell adhesion. These novel data are consistent with PKC and FAK/Src playing an integrated role in integrin activation and integrin-mediated spreading by L. stagnalis haemocytes. We propose a model in which integrin engagement mediates association of PKC with FAK/Src complexes to promote focal adhesion assembly during immune recognition by these cells.

• Publisher: Berlin/Heidelberg : Springer-Verlag,Springer-Verlag,Springer,Springer Nature B.V
• Subject: adhesion / Animals / antagonists & inhibitors / Biomedical and Life Sciences / Biomedicine / cell adhesion / Cell adhesion & migration / Cell Adhesion - drug effects / Cell Movement / Cell Movement - drug effects / confocal microscopy / cytology / drug effects / enzymology / Fibronectins / Fibronectins - pharmacology / Focal adhesion kinase / Focal Adhesion Protein-Tyrosine Kinases / Focal Adhesion Protein-Tyrosine Kinases - metabolism / Focal Adhesions / Focal Adhesions - drug effects / Focal Adhesions - enzymology / Freshwater / Hemocyte/haemocyte / hemocytes / Hemocytes - cytology / Hemocytes - drug effects / Hemocytes - enzymology / Human Genetics / Immune system / immunity / Integrin-mediated cell adhesion / Integrins / Kinases / Lymnaea / Lymnaea - cytology / Lymnaea - drug effects / Lymnaea - enzymology / Lymnaea stagnalis / Lymnaea stagnalis (Mollusca) / metabolism / Models, Biological / Molecular Medicine / Mollusca / Mollusks / non-specific protein-tyrosine kinase / Oligopeptides / Oligopeptides - pharmacology / phagocytes / pharmacology / phosphorylation / Phosphorylation - drug effects / protein kinase C / Protein Kinase C - antagonists & inhibitors / Protein Kinase C - metabolism / Protein Kinase Inhibitors / Protein Kinase Inhibitors - pharmacology / Protein Transport / Protein Transport - drug effects / Proteomics / pseudopodia / Regular Article / scanning electron microscopy / Signal Transduction / Signal Transduction - drug effects / snails / src-Family Kinases / src-Family Kinases - antagonists & inhibitors / src-Family Kinases - metabolism / tyrosine / Western blotting