Gregory J. Pazour

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.

Ror2 signaling regulates Golgi structure and transport through IFT20 for tumor invasiveness

Signaling through the Ror2 receptor tyrosine kinase promotes invadopodia formation for tumor invasion. Here, we identify intraflagellar transport 20 (IFT20) as a new target of this signaling in tumors that lack primary cilia, and find that IFT20 mediates the ability of Ror2 signaling to induce the invasiveness of these tumors. We also find that IFT20 regulates the nucleation of Golgi-derived microtubules by affecting the GM130-AKAP450 complex, which promotes Golgi ribbon formation in achieving polarized secretion for cell migration and invasion. Furthermore, IFT20 promotes the efficiency of transport through the Golgi complex. These findings shed new insights into how Ror2 signaling promotes tumor invasiveness, and also advance the understanding of how Golgi structure and transport can be regulated.

The tiny eukaryote Ostreococcus provides genomic insights into the paradox of plankton speciation

The smallest known eukaryotes, at ≈1-μm diameter, are Ostreococcus tauri and related species of marine phytoplankton. The genome of Ostreococcus lucimarinus has been completed and compared with that of O. tauri. This comparison reveals surprising differences across orthologous chromosomes in the two species from highly syntenic chromosomes in most cases to chromosomes with almost no similarity. Species divergence in these phytoplankton is occurring through multiple mechanisms acting differently on different chromosomes and likely including acquisition of new genes through horizontal gene transfer. We speculate that this latter process may be involved in altering the cell-surface characteristics of each species. In addition, the genome of O. lucimarinus provides insights into the unique metal metabolism of these organisms, which are predicted to have a large number of selenocysteine-containing proteins. Selenoenzymes are more catalytically active than similar enzymes lacking selenium, and thus the cell may require less of that protein. As reported here, selenoenzymes, novel fusion proteins, and loss of some major protein families including ones associated with chromatin are likely important adaptations for achieving a small cell size.

PDGFRαα Signaling Is Regulated through the Primary Cilium in Fibroblasts

Recent findings show that cilia are sensory organelles that display specific receptors and ion channels, which transmit signals from the extracellular environment via the cilium to the cell to control tissue homeostasis and function. Agenesis of primary cilia or mislocation of ciliary signal components affects human pathologies, such as polycystic kidney disease and disorders associated with Bardet-Biedl syndrome. Primary cilia are essential for hedgehog ligand-induced signaling cascade regulating growth and patterning. Here, we show that the primary cilium in fibroblasts plays a critical role in growth control via platelet-derived growth factor receptor alpha (PDGFRalpha), which localizes to the primary cilium during growth arrest in NIH3T3 cells and primary cultures of mouse embryonic fibroblasts. Ligand-dependent activation of PDGFRalphaalpha is followed by activation of Akt and the Mek1/2-Erk1/2 pathways, with Mek1/2 being phosphorylated within the cilium and at the basal body. Fibroblasts derived from Tg737(orpk) mutants fail to form normal cilia and to upregulate the level of PDGFRalpha; PDGF-AA fails to activate PDGFRalphaalpha and the Mek1/2-Erk1/2 pathway. Signaling through PDGFRbeta, which localizes to the plasma membrane, is maintained at comparable levels in wild-type and mutant cells. We propose that ciliary PDGFRalphaalpha signaling is linked to tissue homeostasis and to mitogenic signaling pathways.

A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney

Polycystic kidney disease (PKD) is a common human genetic illness. It is characterized by the formation of multiple kidney cysts that are thought to result from over-proliferation of epithelial cells. Zebrafish larvae can also develop kidney cysts. In an insertional mutagenesis screen in zebrafish, we identified 12 genes that can cause cysts in the glomerular-tubular region when mutated and we cloned 10 of these genes. Two of these genes, vhnf1 (tcf2) and pkd2, are already associated with human cystic kidney diseases. Recently, defects in primary cilia have been linked to PKD. Strikingly, three out of the 10 genes cloned in this screen are homologues of Chlamydomonas genes that encode components of intraflagellar transport (IFT) particles involved in cilia formation. Mutation in a fourth blocks ciliary assembly by an unknown mechanism. These results provide compelling support for the connection between cilia and cystogenesis. Our results also suggest that lesions in genes involved in cilia formation and function are the predominant cause of cystic kidney disease, and that the genes identified here are excellent candidates for novel human PKD genes.

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.

Intraflagellar transport and cilia-dependent diseases

Intraflagellar transport involves the movement of large protein particles along ciliary microtubules and is required for the assembly and maintenance of eukaryotic cilia and flagella. Intraflagellar-transport defects in the mouse cause a range of diseases including polycystic kidney disease, retinal degeneration and the laterality abnormality situs inversus, highlighting the important role that motile, sensory and primary cilia play in vertebrates.

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.