ABSTRACT
For many years, molecular and biochemical studies of mammalian (especially human) respiratory cilia and ciliated cells have been hampered by the relative lack of available material. In contrast, studies of simpler organisms, such as Paramecium and Chlamydomonas, which are easily grown in large quantities in a laboratory setting, have provided a substantial amount of knowledge about the structural organization and function of proteins in the cilia and flagella of these organisms. While many of the basic structural features of cilia have been highly conserved between species, a complete understanding of human respiratory cilia will require detailed studies of human-derived material. Recently, advances in the techniques used to culture human airway epithelial cells have provided researchers with the ability to cultivate increased numbers of ciliated cells. The availability of substantial amounts of high-quality starting material, combined with scientific advances in other areas, including the sequencing of the human genome and the ability to rapidly identify proteins by mass spectrometry, has made possible studies of human cilia that previously were impractical. This chapter will briefly describe our model for culturing human ciliated cells and isolating 127
ciliary axonemes in sufficient quantities for biochemical analysis. An example of a current research project utilizing purified human axonemes will also be presented. THE MODEL
For the majority of our studies, we use human bronchial epithelial (HBE) cells, although cultures derived from nasal epithelial cells are also suitable. Briefly, large airways are excised from excess surgical tissue obtained by Institutional Review Board-approved protocols and digested with protease as described by Bemacki et al. (1). The isolated HBE cells are washed and plated on collagen-coated tissue culture dishes (100 mm diameter) at a density of 1-2 X 106 cells per dish in modified LHC9 medium. Using these conditions, the cells expand as an undifferentiated monolayer and are approximately 75% confluent after 7 days. The cells are collected by trypsinization and plated on collagen coated permeable membranes (Transwell-COL inserts; Costar) at a density of 1-2.5 X 105 cells/ cm2 in ALI medium, a mixture of LHC9 and DMEM that results in improved mucociliary differentiation (1,2). When the cultures reach confluence, usually after about 3 days, the apical medium is removed and the cells are fed basally for the remainder of the culture period. Exposing the apical surface of cultures of airway epithelium to air has been previously shown to significantly enhance ciliated cell differentiation (3,4). During the next 3-4 weeks, the cultures progress
from a single layer of undifferentiated, basal type cells, to a multilayered columnar epithelium containing well-differentiated secretory cells and abundant ciliated cells (Fig. 1). An interesting feature of these well-differentiated cultures is the appearance of small areas where the cilia have become orientated in such a manner that mucociliary transport occurs in a circular pattern. Although clearly different from the unidirectional transport observed in vivo, the ability of these cultures to reproduce the essential features of mucociliary transport in vitro provides further evidence that these cultures provide a good model for studies of cilia and ciliated cells. In addition, cultures that spontaneously develop these areas of mucus transport provide a model for additional studies (5). ISOLATION OF CILIARY AXONEMES Many of the projects underway in our laboratory require the isolation of ciliary axonemes. We have found that the basic procedure developed by Has tie et al.(6) effectively removes ciliary axonemes from our HBE cultures with a minimal amount of contamination. After washing the cultures multiple times with PBS to remove mucus and any accumulated cellular debris, a small volume of buffer containing 0.1% Triton X-100 and 10 mM CaCl2 is added to the surface of the cultures. The culture is gently rocked for approximately 1 minute, the supernatant is collected, and the procedure is repeated. The two washings are pooled and centrifuged at low speed (1000 X g) to pellet cellular debris, and the ciliary axonemes are then collected from the supernatant by centrifugation at 16,000 g. For most studies, the axonemes are washed by resuspension in 0.1% Triton X-100 containing buffer and centrifuged as above. As shown in Figure 2A, this
procedure yields a highly enriched preparation of ciliary axonemes, which when examined at higher magnification (Fig. 2B), demonstrate the characteristic structural features of axonemes, including the 9 + 2 microtubules, inner and outer dynein arms, and radial spokes. The ability to use heavily ciliated cultures of HBE cells eliminates many sources of contamination and variability that are inherent when using in vivo-derived material and certainly contributes to the purity of this axonemal preparation. ANALYSIS OF AXONEMAL PROTEINS
One of the research interests in our laboratory is the identification of mutations that cause primary ciliary dyskinesia (PCD). Primary ciliary dyskinesia is an inherited disease in which defects in the structure of the ciliary axoneme result in impaired or absent mucociliary clearance. Individuals with PCD suffer from recurring infections in the sinuses, middle ear, and respiratory tract. The most commonly reported ciliary defect in PCD, as determined by electron microscopy, is the absence of outer dynein arms, although many other abnormalities have been reported and the disease is believed to be heterogeneous. Because the defects in ciliary axonemes frequently involve the absence of protein structures, we reasoned that a careful comparison of proteins present in purified axonemes from normal and PCD individuals might allow us to identify proteins missing in PCD patients. Proteins that are missing in the axonemes from PCD patients would be excellent candidates for further analysis to determine if the gene coding for the protein contained a causative mutation. This approach is similar to that used to identify the mutated protein in radial spoke mutants of Chlamydomonas (7,8).To maximize the sensitivity of our analysis, HBE cells obtained from normal individuals or a patient with PCD were cultured in the presence of 35S-labeled methionine. Ciliary axonemes were isolated as described above and equal amounts of radioactive proteins from each sample were separated by two-dimensional gel electrophoresis. Preliminary experiments, which compared several normal samples, demonstrated that the pattern of axonemal proteins is highly reproducible. However, our initial comparisons between axonemes isolated from normal and PCD cells (Fig. 3) revealed no obvious differences. Currently we are exploring the use of narrow range pH strips for the first dimension to increase the resolution of the axonemal proteins and improve the detection of missing or altered proteins. We have also begun to analyze the high molecular weight dynein heavy chains, which do not focus on 2-D gels, by SDS-PAGE. Proteins that are missing or altered in the axonemes of PCD patients will be identified by using mass spectrometry and searching of databases. In addition, a number of proteins from different regions of the 2-D gel pattern of normal axonemes will be identified by the same technique. These proteins will provide reference points so that additional samples can be easily compared.
