Methods for Studying Ciliary Import Mechanisms
Cilia and flagella are microtubule-based organelles that play important roles in human health by contributing to cellular motility as well as sensing and responding to environmental cues. Defects in cilia formation and function cause a broad class of human genetic diseases called ciliopathies. To carry out their specialized functions, cilia contain a unique complement of proteins that must be imported into the ciliary compartment. In this chapter, we describe methods to measure the permeability barrier of the ciliary gate by microinjection of fluorescent proteins and dextrans of different sizes into ciliated cells. We also describe a fluorescence recovery after photobleaching (FRAP) assay to measure the entry of ciliary proteins into the ciliary compartment. These assays can be used to determine the molecular mechanisms that regulate the formation and function of cilia in mammalian cells.
Key wordsCilia Nuclear import Ciliary import Microinjection FRAP
We thank members of the Verhey Lab for advice and discussions. We are grateful to Steve Lentz and the Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center (funded by NIDDK under NIH5P60 DK20572) for help with confocal imaging. Work in K.J. Verhey Lab is supported by NIGMS of the NIH under award numbers R01GM070862 and R01GM116204.