A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway.
The signalling pathway for the hepatocyte growth factor receptor, Met/HGF-R, is hijacked by the bacterial surface protein InlB to induce Listeria monocytogenes entry into non-phagocytic cells. We previously showed that Listeria invades host cells by interacting with specialized microdomains of the host plasma membrane called lipid rafts. In this study, we analysed in living cells signalling events that are crucial for Listeria entry using a fluorescence resonance energy transfer-based microscopic method. Phosphoinositide (PI) 3-kinase activity and Rac1 signalling induced by Listeria interacting with epithelial cells were monitored as well as signalling induced by soluble InlB and the Met natural ligand HGF. We found that InlB and HGF induced similar kinetics of PI 3-kinase and Rac1 activation. PI 3-kinase activation was upstream and independent of Rac1 activation. Cholesterol-depletion experiments were performed to address the role of lipid rafts in Met signalling. The amount of 3'-phosphoinositides produced by PI 3-kinase was not affected by cholesterol depletion, while their membrane dynamic was cholesterol-dependent. Rac1 activation, downstream from PI 3-kinase, was cholesterol-dependent suggesting that the spatial distribution of 3'-phosphoinositides within membrane microdomains is critical for Rac1 activation and consequently for F-actin assembly at bacterial entry site.
Seveau
Stéphanie
S
Institute Pasteur, Unité des Interactions Bactéries-Cellules, Paris, France.
Tham
To N
TN
Payrastre
Bernard
B
Hoppe
Adam D
AD
Swanson
Joel A
JA
Cossart
Pascale
P
D000818:Animals; D001426:Bacterial Proteins / Q000502:physiology*; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000378:metabolism* / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007700:Kinetics; D008089:Listeria monocytogenes / Q000502:physiology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism / Q000382:microbiology; D008565:Membrane Proteins / Q000502:physiology*; D008856:Microscopy, Fluorescence; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D019859:Proto-Oncogene Proteins c-met / Q000502:physiology*; D015398:Signal Transduction; D014162:Transfection; D014709:Vero Cells; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D001426:Bacterial Proteins; D002784:Cholesterol; D008164:Luminescent Proteins; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases; D019859:Proto-Oncogene Proteins c-met; D020830:rac1 GTP-Binding Protein; C095768:inlB protein, Listeria monocytogenes