Detailed proteomic analyses of mammalian olfactory and rod photoreceptor sensory cilia are now available, providing an inventory of resident ciliary proteins and laying the foundation for future studies of developmental and spatio-temporal changes in the composition of sensory cilia. Cilia purification methods that were elaborated and perfected over several decades were essential for these advances. In contrast, the proteome of primary cilia is yet to be established, because purification procedures for this organelle have been developed only recently. In this chapter we review current techniques for the purification of olfactory and photoreceptor cilia, and evaluate methods designed for the selective isolation of primary cilia.

In spite of their complete lack of any structural features that characterize membrane proteins, cytosolic nucleoside-diphosphate kinases (NDPKs) have been found repeatedly to associate with membranes. In some instances the recruitment of cytosolic NDPKs to membranes was attributed to interactions with peripheral or integral membrane proteins, but in many cases the mechanism underlying the association of NDPKs with membranes remained unknown. We show here that cytosolic NDPKs bind directly to membrane lipids in a dynamic process that is controlled by its substrates, nucleoside tri- and diphosphates, and can be fully reconstituted with chemically defined, protein-free phospholipids and recombinant NDPK, or with purified NDPK. Our results uncover a novel mechanism for the reversible targeting of soluble NDPKs to membranes, where they may act as a reservoir of high energy phosphate, supporting the operation of membrane-based processes that utilize nucleotides other than ATP, such as intracellular traffic and phospholipid biosynthesis.

Inmunofluorescence staining of murine NIH3T3 fibroblasts grown at high density shows that conventional nucleoside diphosphate (NDP) kinases A and B localize to a sensory organelle, the primary cilium. Similar results are obtained with Xenopus A6 kidney epithelial cells, suggesting that NDP kinases are a universal component of the primary cilium. The translocation of NDP kinase into primary cilia depends on size, taking place only when cilia reach a critical length of 5-6 μm. In mature cilia, NDP kinases are distributed along the ciliary shaft in a punctate pattern that is distinct from the continuous staining observed with acetylated α-tubulin, a ciliary marker and axonemal component. Isolation of a fraction enriched in primary cilia from A6 cells led to the finding that ciliary NDP kinase is enzymatically active, and is associated with the membrane and the matrix, but not the axoneme. In contrast, acetylated α-tubulin is found in the axoneme and to a lesser extent, in the membrane. Based on the tightly regulated translocation process and the subciliary distribution pattern of NDP kinase, we propose that it plays a role in the elongation and maintenance of primary cilia by its ability to regenerate the GTP utilized by ciliary microtubule turnover and transmembrane signaling.

We used immunofluorescence techniques to determine the localization of nucleoside diphosphate (NDP) kinase in NIH-3T3 fibroblasts. We found that cytoplasmic NDP kinase can be separated into two populations according to subcellular localization and response to extracellular stimuli. Specifically, within minutes of stimulation of resting fibroblasts with serum, growth factors or bombesin, a portion of NDP kinase becomes associated with membrane ruffles and lamellipodia. Another pool of NDP kinase accumulates independently of stimulation around intracellular vesicles. Transfection of cells with activated Rac mimics, whereas expression of dominant negative Rac inhibits, the effects of extracellular stimulation on the translocation of NDP kinase to the cell cortex. Neither Rac mutant affects the vesicle-associated pool. Association of NDP kinase with vesicles depends on microtubule integrity and is disrupted by nocodazole. In cell-free assays NDP kinase binds tightly to membrane vesicles associated with taxol-stabilized microtubules. Binding of NDP kinase to this fraction is reduced by ATP and abolished by GTP, as well as guanine nucleotides that are NDP kinase substrates. Thus, the localization of the two NDP kinase pools identified here is regulated independently by distinct cellular components: the appearance of cortical NDP kinase is a consequence of Rac activation, whereas vesicular NDP kinase is responsive to microtubule dynamics and nucleotides, in particular GTP. These results suggest that in fibroblasts NDP kinase participates in Racrelated cortical events and in GTP-dependent processes linked to intracellular vesicle trafficking.

Cytosolic nucleoside diphosphate kinases (NDPKs) have been implicated in a variety of signaling pathways that occur at membranes, including those that control cell migration and spreading. This is particularly intriguing, as cytosolic NDPKs (NDPK A and NDPK B) are soluble proteins and do not have membrane-binding motifs, leading to the question: how do NDPK's participate in such a wide array of membrane signaling processes?

Our lab has shown that one portion of cytosolic NDPK is translocated to the ruffling plasma membrane upon activation of both receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) and that the Rac1 signaling pathway is responsible for that migration. Although NDPK does not bind directly to Rac1, it moves to the cell periphery in conjunction with Rac1. While investigating the association of cytosolic NDPK with the plasma membrane, we found that another pool of NDPK is bound to membrane vesicles that are associated with microtubules (Mt/Ves). A detailed study of this NDPK population shows that, unlike the pool that is involved in Rac1 signaling, NDPK's presence in these vesicles is not dependent on extracellular stimulation; rather, it is controlled by the nucleotide triphosphate to nucleotide diphosphate ratio ([NTP]/[NDP]), as evidenced by the effect of nucleotides on Mt/Ves isolated from fibroblasts. More importantly, purified and cytosolic NDPKs bind to both immobilized lipids and liposomes in a nucleotide-sensitive manner. This indicates that NDPK can bind directly to intracellular membrane compartments, most likely to provide CTP for phospholipid biosynthesis and GTP for the many small GTPases involved in microtubule-dependent traffic.

We also found that NDPK localizes to yet another microtubule-based cell compartment: the sensory primary cilium, an organelle implicated in many signaling pathways. NDPK enters the cilium during its development, when it reaches about 5.5 microns (or 24% of final primary cilia length) in A6 cells. In primary cilia NDPK is present in the soluble portion, or matrix, and in association with the membrane fraction. The function of NDPK within primary cilia is most likely to regenerate GTP for microtubule turnover and for signaling systems, making it an important contributor to primary cilia structure and function.