Subcellular Analysis of the Motor Protein Apparatus in Ependymal Cilia
Aims: Cilia are evolutionary conserved, hair-like organelles protruding from the surface of almost all human cells. Their function is pleiotropic and involves mucociliary clearance of the airways, determination of left–right body asymmetry, sperm, and oviductal propulsion as well as generation of a directional cerebrospinal fluid flow. Dysfunction of motile ependymal cilia inevitably leads to internal hydrocephalus in mouse models whereas in humans development of hydrocephalus is facultative. High-resolution immunofluorescence microscopy allows the visualization of specific proteins and their distribution within the cilium. The analysis of ependymal cilia by IF has concentrated on murine models of hydrocephalus development so far. The aim of this study is to compare the motor protein apparatus in cilia of human respiratory and ependymal cells.
Methods: IF analysis of human ependymal tissue cryosections with antibodies directed against components of dynein arms, nexin links, and radial spokes was performed according to an established protocol. Images were acquired with a Zeiss Apotome Axiovert 200 camera and processed with AxioVision 4.8 and Adobe Illustrator.
Results: Analogous to cilia in respiratory epithelia, human ependymal cilia contain two types of outer dynein arms located in the proximal respectively distal axoneme. The radial spoke protein RSPH4A and the nexin link component GAS8 are colocalized with acetylated α-tubulin und thus present along the entire length of the axoneme.
Conclusion: The localization of selected proteins (RSPH4A and GAS8) is shown in human ependymal cilia for the first time. Human respiratory and ependymal cilia demonstrate an identical immunolocalization of antigens analyzed in dynein arms, radial spokes, and nexin links.
Keywords: ependymal, cilia, immunofluorescence, hydrocephalus, dynein, nexin-links, radial spokes.