Chapter Three - Analysis of Primary Cilia in Directional Cell Migration in Fibroblasts
Introduction
The primary cilium is a microtubule-based, solitary organelle that emanates from the centrosomal mother centriole during growth arrest of most cell types of the human body to coordinate signaling pathways that critically regulate cellular processes during development and in tissue homeostasis (Christensen et al., 2012, Satir and Christensen, 2007). Consequently, defects in ciliary formation or compartmentalization of ciliary receptors and downstream components in signal transduction lead to a series of pathologies, now known as ciliopathies (Hildebrandt et al., 2011, Waters and Beales, 2011). Here, we describe methods in analyzing ciliary function in directional cell migration in fibroblasts, which when defective causes developmental disorders and is implicated in diseases such as fibrosis, tumorigenesis, and cancer cell invasion.
Recognition of the relationship of primary cilia to fibroblast migration antedates the modern era of quantitative study. The fibroblast primary cilium was described by Sorokin in the early 1960s (Sorokin, 1962), and Tucker and coworkers studied its formation during growth arrest and relationship to the cell cycle (Tucker, Pardee, & Fujiwara, 1979). Several investigators, notably Albrecht-Buehler, noted that the primary cilium points in the direction of cell migration (Albrecht-Buehler, 1977). However, analysis of the ciliary signaling pathways in cell migration has only been achieved in the past decade (Christensen et al., 2008, Jones et al., 2012, Lu et al., 2008), particularly with the realization that growth arrest-specific proteins, especially platelet-derived growth factor receptor alpha (PDGFRα), were associated with the pathway in lamellipodia formation and directional cell migration (Schneider et al., 2005, Schneider et al., 2009, Schneider et al., 2010). As outlined in this chapter, major advances in immunomicroscopy, scratch assay, and micropipette analysis have contributed to our understanding of primary cilia in cell migration.
The role of primary ciliary in PDGFRα signaling and cell migration began with cultures of NIH3T3 fibroblasts, but an important tool for analysis are mouse embryonic fibroblasts (MEFs) derived from either Tg737orpk mice or their wild-type (wt) littermates. Serum deprivation in NIH3T3 or wt MEFs leads to the formation of primary cilia and upregulation of PDGFRα, while Tg737orpk mutant MEFs function as controls where neither event occurs (Schneider et al., 2005). PDGF-AA is a specific ligand for PDGFRαα. A combination of Western blot and immunolocalization experiments using this ligand shows that PDGFRα is transported to and imported into the growing cilium where it dimerizes, becomes phosphorylated, and signals via the AKT and MEK1/2–ERK1/2 pathways to control directional cell migration by influencing the transport and positioning of an Na+/H+ exchange protein to the lamellipodium (Schneider et al., 2005, Schneider et al., 2009, Schneider et al., 2010, Clement et al., 2012). While the use of mutants to define the signal transduction pathways is extremely useful, the system can also be probed using ciliary knockout procedures, RNAi or inhibitors. Since little is actually known about the way primary cilia control cellular events or cytoskeletal organization, it is probable that variations of these techniques will prove useful in not only following cellular changes in other primary cilia signaling systems, certainly where cell migration is involved, but also more generally in delineating pathways from the cilia itself into the cytoplasm and nucleus. In this chapter, with the fibroblast system as a model, we provide protocols for techniques to localize signaling proteins and to measure directional cell migration that is dependent upon primary cilium signaling.
Section snippets
Fibroblast cultures and immunofluorescence microscopy analysis
NIH3T3 cells and MEFs form primary cilia at a frequency of 70–90% in cell cultures that are deprived of serum to induce growth arrest. Maximum ciliation usually occurs by 24–48 h of serum starvation. The cilia are detected by immunofluorescence microscopy (IFM) with antibodies against acetylated (Ac-tub) or glutamylated α-tubulin (Glu-tub), which are posttranslational modification enriched in primary cilia (Pedersen, Schroder, Satir, & Christensen, 2012). The basal body of primary cilia is
Setting up the scratch assay
The scratch assay is a simple method and useful tool for analysis of cell migration in two dimensions (Nobes & Hall, 1999). Although fibroblasts in vivo rarely migrate in a plane, this assay benefits from being relatively easy to monitor and allows for investigation of cell behavior and ligand application under tightly controlled conditions. Differences between migration assays in one, two, or three dimensions have been discussed in Baker and Chen (2012) and Cukierman, Pankov, Stevens, and
Setting up the micropipette assay
The goal of the micropipette assay is to provide a localized source of diffusible molecules, to image cells' reaction to the chemical, and to measure or diagram responses. This method has been characterized for use with yeast orienting toward a source of mating factor (Segall, 1993), Dictyostelium streaming toward cyclic AMP (Segall & Gerisch, 1989), macrophage crawling to CSF, metastatic carcinomas (Bailly, Yan, Whitesides, Condeelis, & Segall, 1998), and with MEFs with primary cilia orienting
Summary
The methods described above for (1) immunofluorescence localization of primary cilia in fibroblast cultures; (2) their orientation with respect to the cell axis during directional migration; (3) setting up and utilizing scratch assays to measure directional cell migration in wound healing; (4) studying cell signaling and response with micropipette assays; (5) live-cell imaging; and (6) analysis of individual cell and population migration parameters, have all been successfully tested. Figure 3.1
Acknowledgments
This work was supported by The Lundbeck Foundation, including a visiting Professorship to P. S., The Danish National Science Research Council, and the Novo Foundation (S. T. C.).
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