3 SUMMARYInmunofluorescence 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.4

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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.

The cell's primary cilium is both a mechanical and chemical sensor involved in many signaling pathways. In order to ascertain protein enrichment in the primary cilium or study sub‐ciliary localization of various proteins, it is advantageous to remove the primary cilium from the cell body. The protocol described here gives detailed instructions on purifying primary cilia by separating them from the cell body using shear force. This simple technique avoids using harsh purification conditions that may affect signaling proteins in the cilium or cause the ciliary membrane to disintegrate. In addition, as the cell body remains mostly intact, contamination of the isolated cilia by proteins from the cell body is minimized. This protocol is ideally suited for isolating cilia from renal cell lines, as primary cilia in these cells grow to greater lengths than in other cell types (up to 50‐µm long in Xenopus A6 toad kidney cells as opposed to 1 to 5 µm in NIH3T3 fibroblast cells). Curr. Protoc. Cell Biol. 59:3.42.1‐3.42.9. © 2013 by John Wiley & Sons, Inc.

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