Use of pHluorin to assess the dynamics of axon guidance receptors in cell culture and in the chick embryo.
During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.
University Claude Bernard Lyon1, CGphiMC UMR CNRS 5534, University of Lyon.
Céline
C
Delloye-Bourgeois
Arnaud
A
Jacquier
Julien
J
Falk
Valérie
V
Castellani
D000818:Animals; D001369:Axons / Q000502:physiology*; D019556:COS Cells; D015815:Cell Adhesion Molecules / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D018274:Electroporation; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009419:Nerve Tissue Proteins / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000502:physiology*; D013116:Spinal Cord / Q000502:physiology*
D015815:Cell Adhesion Molecules; D009419:Nerve Tissue Proteins; C485187:PHluorin; D011956:Receptors, Cell Surface; C091529:plexin; D049452:Green Fluorescent Proteins