Immunoelectron microscopy of vesicle transport to the primary cilium of photoreceptor cells

Abstract

Cilia are organelles of high structural complexity. Since the biosynthetic machinery is absent from cilia all their molecular components must be synthesized in organelles of the cytoplasm and subsequently transported to the cilium. Ciliary cargos are thought to be translocated in the membrane of transport vesicles or association with these vesicles to the base of the cilium where the vesicles fuse with the periciliary target membrane for further delivery of their cargo into the ciliary compartment by the intraflagellar transport (IFT). Here we describe a modified preembedding labeling method as an alternative technique to conventional postembedding methods eligible for analyses of ciliary cargo vesicles and the distribution of ciliary molecules in subciliary compartments for immunoelectron microscopy. The preembedding labeling method preserves the antigenicity of ciliary antigens and its application reveals differential localization of individual IFT proteins in vertebrate photoreceptor cilia. Since membrane vesicles are conserved, the preembedding protocol additionally allows the identification of ciliary cargo vesicles by immunolabeling of individual IFT proteins and ciliary targeting molecules in ciliary photoreceptor cells. These results do not only confirm the central function of IFT molecules in ciliary transport, but further strengthen their role in transport processes in the cytoplasm. Furthermore, evidence for different alternative transport routes of cargo vesicles directed to different target membranes is gathered.