Imaging centrosomes and cilia in the mouse kidney.
The centrosome and cilium are evolutionarily conserved components of the microtubule cytoskeleton, and act as a cellular signaling center that regulates the activity of numerous developmental signaling pathways. Several genetic syndromes, called the ciliopathies, are associated with defects in the structure or function of the centrosome-cilium complex. In the mammalian kidney, these organelles are found at the apical surface of renal epithelial cells lining the various segments of the nephron, where they relay information from the extracellular environment to the interior of the cell. Cilium-based signaling plays an important role in the development and homeostasis of mammalian kidneys, and ciliary dysfunction is implicated in the pathogenesis of cystic kidney disease. Given the importance of centrosomes and cilia in renal function, techniques used to visualize these organelles, analyze their composition, and test their functionality have become essential in many studies of kidney development and disease. Fluorescence microscopy is a powerful, widely used technique that has enhanced our understanding of molecular mechanisms that regulate the assembly, maintenance, and function of these organelles in various organs. Here, we present detailed steps for the isolation of kidneys from adult and embryonic mice, describe protocols to label centrosomes and cilia in renal tissues, and methods used to culture and image kidneys ex vivo.