An NGF-induced Exo70-TC10 complex locally antagonises Cdc42-mediated activation of N-WASP to modulate neurite outgrowth.
NGF-induced differentiation of PC12 cells is mediated by actin-polymerisation-driven membrane protrusion, involving GTPase signalling pathways that activate actin nucleation promoting factors such as the neural Wiskott-Aldrich syndrome protein (N-WASP). Expression of the exocyst subunit Exo70 in PC12 cells and neurons leads to the generation of numerous membrane protrusions, an effect that is strongly potentiated upon NGF-induced differentiation. Förster resonance energy transfer (FRET) imaging by fluorescence lifetime microscopy (FLIM) reveals an NGF-induced interaction of activated TC10 with Exo70. Expression of dominant-negative mutants and siRNA-mediated knockdown implicates N-WASP in NGF-induced Exo70-TC10-mediated membrane protrusion. However, FRET imaging of N-WASP activation levels of cells expressing Exo70 and/or constitutively active TC10 reveals that this complex locally antagonises the NGF-induced activation of N-WASP in membrane protrusions. Experiments involving siRNA-mediated knockdown of Cdc42 and overexpression of constitutively active Cdc42 confirm that the Exo70-TC10 complex mainly targets the NGF-induced Cdc42-dependent activation of N-WASP. Our results show that Exo70 is responsible for the correct targeting of the Exo70-TC10 complex to sites of membrane protrusion. The functional uncoupling between both pathways represents a novel regulatory mechanism that enables switching between morphologically distinct - Cdc42- or TC10-dominated - forms of cellular membrane outgrowth.
Pommereit
Dagmar
D
European Neuroscience Institute-Göttingen, Cell Biophysics Group and DFG Research Center for Molecular Physiology of the Brain (CMPB), D-37073 Göttingen, Germany.
Wouters
Fred S
FS
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D020558:GTP Phosphohydrolases / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D051379:Mice; D020932:Nerve Growth Factor / Q000378:metabolism*; D016501:Neurites / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
C109901:Exoc7 protein, rat; D033921:Vesicular Transport Proteins; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D020932:Nerve Growth Factor; D020558:GTP Phosphohydrolases; C503262:Rhoq protein, rat; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins