Mapping the Localization of Proteins Within Filopodia Using FiloMap

Filopodia are fingerlike membrane protrusions extended by cells to sense their environment. Filopodia are widely used by migrating cells in vivo and directly contribute to several physiological processes and diseases. Due to the essential roles of filopodia in sensing the extracellular environment, there is a need to characterize their composition and ultrastructure further. This chapter highlights FiloMap, an image analysis pipeline that utilizes Fiji and R to map the localization of proteins within filopodia from microscopy images. I provide step-by-step protocols on (a) setting up FiloMap in Fiji and R, (b) extracting line intensity profiles from filopodia stainings in Fiji, (c) further analyzing line intensity profiles in R, and (d) creating filopodia maps to compare the localization of multiple proteins within filopodia. Notably, while FiloMap was written to analyze filopodia, the analysis pipeline described here can also analyze and compile any line intensity profiles.