George B. Witman

Proteomic analysis of a eukaryotic cilium

Cilia and flagella are widespread cell organelles that have been highly conserved throughout evolution and play important roles in motility, sensory perception, and the life cycles of eukaryotes ranging from protists to humans. Despite the ubiquity and importance of these organelles, their composition is not well known. Here we use mass spectrometry to identify proteins in purified flagella from the green alga Chlamydomonas reinhardtii. 360 proteins were identified with high confidence, and 292 more with moderate confidence. 97 out of 101 previously known flagellar proteins were found, indicating that this is a very complete dataset. The flagellar proteome is rich in motor and signal transduction components, and contains numerous proteins with homologues associated with diseases such as cystic kidney disease, male sterility, and hydrocephalus in humans and model vertebrates. The flagellum also contains many proteins that are conserved in humans but have not been previously characterized in any organism. The results indicate that flagella are far more complex than previously estimated.

Polycystin-2 localizes to kidney cilia and the ciliary level is elevated in orpk mice with polycystic kidney disease

We thank Dr J. Lawrence and members of her laboratory for microscope time and assistance, Dr P. Furcinitti for assistance, and Drs Y. Cai and S. Somlo for the YCC2 antibody and for critically reading this manuscript. This work was supported by NIH GM60992 (GJP), GM30626 (GBW), GM14642 (JLR), and by the Robert W. Booth Fund at the Greater Worcester Community Foundation (GBW).

The vertebrate primary cilium is a sensory organelle

The primary cilium is a generally non-motile cilium that occurs singly on most cells in the vertebrate body. The function of this organelle, which has been the subject of much speculation but little experimentation, has been unknown. Recent findings reveal that the primary cilium is an antenna displaying specific receptors and relaying signals from these receptors to the cell body. For example, kidney primary cilia display polycystin-2, which forms part of a Ca2+ channel that initiates a signal that controls cell differentiation and proliferation. Kidney primary cilia also are mechanosensors that, when bent, initiate a Ca2+ signal that spreads throughout the cell and to neighboring cells. Primary cilia on other cell types specifically display different receptors, including those for somatostatin and serotonin.

The intraflagellar transport protein, IFT88, is essential for vertebrate photoreceptor assembly and maintenance

Approximately 10% of the photoreceptor outer segment (OS) is turned over each day, requiring large amounts of lipid and protein to be moved from the inner segment to the OS. Defects in intraphotoreceptor transport can lead to retinal degeneration and blindness. The transport mechanisms are unknown, but because the OS is a modified cilium, intraflagellar transport (IFT) is a candidate mechanism. IFT involves movement of large protein complexes along ciliary microtubules and is required for assembly and maintenance of cilia. We show that IFT particle proteins are localized to photoreceptor connecting cilia. We further find that mice with a mutation in the IFT particle protein gene, Tg737/IFT88, have abnormal OS development and retinal degeneration. Thus, IFT is important for assembly and maintenance of the vertebrate OS.

CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content

Entry and exit of proteins into flagella is gauged by CEP290 in the transition zone.

The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella

The Bardet-Biedl syndrome protein complex (BBSome) is a cargo adapter rather than an essential part of the intraflagellar transport (IFT) machinery.

Cytoplasmic dynein nomenclature

A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms.

Mutations in Hydin impair ciliary motility in mice

Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility, and mice with Hydin defects develop lethal hydrocephalus. To determine if defects in Hydin cause hydrocephalus through a mechanism involving cilia, we compared the morphology, ultrastructure, and activity of cilia in wild-type and hydin mutant mice strains. The length and density of cilia in the brains of mutant animals is normal. The ciliary axoneme is normal with respect to the 9 + 2 microtubules, dynein arms, and radial spokes but one of the two central microtubules lacks a specific projection. The hydin mutant cilia are unable to bend normally, ciliary beat frequency is reduced, and the cilia tend to stall. As a result, these cilia are incapable of generating fluid flow. Similar defects are observed for cilia in trachea. We conclude that hydrocephalus in hydin mutants is caused by a central pair defect impairing ciliary motility and fluid transport in the brain.

The Autosomal Recessive Polycystic Kidney Disease Protein Is Localized to Primary Cilia, with Concentration in the Basal Body Area

Recent evidence suggests that structural and functional abnormalities of primary cilia in kidney epithelia are associated with mouse and human autosomal dominant polycystic kidney disease. To determine whether fibrocystin/polyductin/tigmin (FPC), the protein product encoded by the PKHD1 gene that is responsible for autosomal recessive polycystic kidney disease among human subjects, is also a component of primary cilia in the kidney, antipeptide antibodies to the carboxyl-terminal intracellular domain and amino-terminal extracellular domain of FPC were generated and were characterized with immunoblotting and immuno-light and -electron microscopy. Immunolocalization in normal kidney tissue sections and cultured kidney cells demonstrated that FPC was localized to the primary cilia and concentrated on the basal bodies in both kidney tissue sections and cultured kidney cells. The FPC expression pattern was not altered in kidney cells with Pkd1 mutations. These findings suggest that FPC is a functional and/or structural component of primary cilia in kidney tubular cells. It is proposed that the pathogenesis of autosomal recessive polycystic kidney disease is linked to the dysfunction of primary cilia.

Pericentrin forms a complex with intraflagellar transport proteins and polycystin-2 and is required for primary cilia assembly.

Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases. Here, we show that the centrosome protein pericentrin (Pcnt) colocalizes with IFT proteins to the base of primary and motile cilia. Immunogold electron microscopy demonstrates that Pcnt is on or near basal bodies at the base of cilia. Pcnt depletion by RNA interference disrupts basal body localization of IFT proteins and the cation channel polycystin-2 (PC2), and inhibits primary cilia assembly in human epithelial cells. Conversely, silencing of IFT20 mislocalizes Pcnt from basal bodies and inhibits primary cilia assembly. Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions. Pcnt antibodies coimmunoprecipitate IFT proteins and PC2 from several cell lines and tissues. We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.