Analysis of soluble protein entry into primary cilia using semipermeabilized cells

Abstract

The primary cilium is a protrusion from the cell surface that serves as a specialized compartment for signal transduction. Many signaling factors are known to be dynamically concentrated within cilia and to require cilia for their function. Yet protein entry into primary cilia remains poorly understood. To enable a mechanistic analysis of soluble protein entry into cilia, we developed a method for semipermeabilization of mammalian cells in which the plasma membrane is permeabilized while the ciliary membrane remains intact. Using semipermeabilized cells as the basis for an in vitro diffusion-to-capture assay, we uncovered a size-dependent diffusion barrier that restricts soluble protein exchange between the cytosol and the cilium. The manipulability of this in vitro system enabled an extensive characterization of the ciliary diffusion barrier and led us to show that the barrier is mechanistically distinct from those at the axon initial segment and the nuclear pore complex. Because semipermeabilized cells enable a range of experimental perturbations that would not be easily feasible in intact cells, we believe this methodology will provide a unique resource for investigating primary cilium function in development and disease.

Keywords: Cilium; Diffusion barrier; Digitonin; Primary cilia; Semipermeabilization.