Tracking Differential Endocytosis and Trafficking of Semaphorin Receptor Complexes in Responding Nerve Growth Cones

The study of receptor endocytosis is important to our understanding of the signal transduction events initiated by axon guidance cues in growth cones. Fab fragments of antibodies to guidance receptors and endocytic cargoes like transferrin and cholera toxin-B are the tools of choice for studying the dynamics of receptor internalization and intracellular trafficking by different pathways. We describe a method where in vitro cultures of growth cones are incubated with these ligands in the presence or absence of Sema3A, followed by stripping of remaining ligand on cell-surface and analysis by immunofluorescence techniques. These techniques can be employed for studying the endocytosis of any axon guidance receptor in response to attractive or repulsive guidance cues and, in particular, to allow the differential trafficking of specific receptor components to be revealed.