Assays to Examine Transmembrane Semaphorin Function In Vitro

Abstract

The semaphorins are a large family of secreted and membrane associated proteins that play numerous key roles in the development and function of the nervous system and other tissues. They have been primarily associated with their function as guidance cues in the developing nervous system. In general, semaphorins have been shown to function as inhibitory guidance cues; however there are also numerous examples where they can function as attractive or permissive cues. Thus it is important to employ a variety of assays to test for semaphorin function. While numerous assays have been established for secreted semaphorins, testing the function of transmembrane semaphorins has been challenging. In this chapter we outline two assays that we have used extensively to test their function. In one assay we examine the effect of a constant source of a transmembrane semaphorin on neurite outgrowth and in a second assay we examine whether neurons will actively avoid growing across islands of cells expressing a transmembrane semaphorin. We have found both assays to be relatively easy to perform and useful to test semaphorin function and signaling.