Jovenal T. San Agustin

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).

Deletion of IFT20 in the mouse kidney causes misorientation of the mitotic spindle and cystic kidney disease

Primary cilia project from the surface of most vertebrate cells and are thought to be sensory organelles. Defects in primary cilia lead to cystic kidney disease, although the ciliary mechanisms that promote and maintain normal renal function remain incompletely understood. In this work, we generated a floxed allele of the ciliary assembly gene Ift20. Deleting this gene specifically in kidney collecting duct cells prevents cilia formation and promotes rapid postnatal cystic expansion of the kidney. Dividing collecting duct cells in early stages of cyst formation fail to properly orient their mitotic spindles along the tubule, whereas nondividing cells improperly position their centrosomes. At later stages, cells lacking cilia have increased canonical Wnt signaling and increased rates of proliferation. Thus, IFT20 functions to couple extracellular events to cell proliferation and differentiation.

Global genetic analysis in mice unveils central role for cilia in congenital heart disease

Congenital heart disease (CHD) is the most prevalent birth defect, affecting nearly 1% of live births; the incidence of CHD is up to tenfold higher in human fetuses. A genetic contribution is strongly suggested by the association of CHD with chromosome abnormalities and high recurrence risk. Here we report findings from a recessive forward genetic screen in fetal mice, showing that cilia and cilia-transduced cell signalling have important roles in the pathogenesis of CHD. The cilium is an evolutionarily conserved organelle projecting from the cell surface with essential roles in diverse cellular processes. Using echocardiography, we ultrasound scanned 87,355 chemically mutagenized C57BL/6J fetal mice and recovered 218 CHD mouse models. Whole-exome sequencing identified 91 recessive CHD mutations in 61 genes. This included 34 cilia-related genes, 16 genes involved in cilia-transduced cell signalling, and 10 genes regulating vesicular trafficking, a pathway important for ciliogenesis and cell signalling. Surprisingly, many CHD genes encoded interacting proteins, suggesting that an interactome protein network may provide a larger genomic context for CHD pathogenesis. These findings provide novel insights into the potential Mendelian genetic contribution to CHD in the fetal population, a segment of the human population not well studied. We note that the pathways identified show overlap with CHD candidate genes recovered in CHD patients, suggesting that they may have relevance to the more complex genetics of CHD overall. These CHD mouse models and >8,000 incidental mutations have been sperm archived, creating a rich public resource for human disease modelling.

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.

The Golgin GMAP210/TRIP11 anchors IFT20 to the Golgi complex

Eukaryotic cells often use proteins localized to the ciliary membrane to monitor the extracellular environment. The mechanism by which proteins are sorted, specifically to this subdomain of the plasma membrane, is almost completely unknown. Previously, we showed that the IFT20 subunit of the intraflagellar transport particle is localized to the Golgi complex, in addition to the cilium and centrosome, and hypothesized that the Golgi pool of IFT20 plays a role in sorting proteins to the ciliary membrane. Here, we show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP210/Trip11. Mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction, ventricular septal defects of the heart, omphalocele, and lung hypoplasia. Cells lacking GMAP210 have normal Golgi structure, but IFT20 is no longer localized to this organelle. GMAP210 is not absolutely required for ciliary assembly, but cilia on GMAP210 mutant cells are shorter than normal and have reduced amounts of the membrane protein polycystin-2 localized to them. This work suggests that GMAP210 and IFT20 function together at the Golgi in the sorting or transport of proteins destined for the ciliary membrane.

IFT25 links the signal-dependent movement of Hedgehog components to intraflagellar transport.

The intraflagellar transport (IFT) system is required for building primary cilia, sensory organelles that cells use to respond to their environment. IFT particles are composed of about 20 proteins, and these proteins are highly conserved across ciliated species. IFT25, however, is absent from some ciliated organisms, suggesting that it may have a unique role distinct from ciliogenesis. Here, we generate an Ift25 null mouse and show that IFT25 is not required for ciliary assembly but is required for proper Hedgehog signaling, which in mammals occurs within cilia. Mutant mice die at birth with multiple phenotypes, indicative of Hedgehog signaling dysfunction. Cilia lacking IFT25 have defects in the signal-dependent transport of multiple Hedgehog components including Patched-1, Smoothened, and Gli2, and fail to activate the pathway upon stimulation. Thus, IFT function is not restricted to building cilia where signaling occurs, but also plays a separable role in signal transduction events.

IFT27 Links the BBSome to IFT for Maintenance of the Ciliary Signaling Compartment

Vertebrate hedgehog signaling is coordinated by the differential localization of the receptors patched-1 and Smoothened in the primary cilium. Cilia assembly is mediated by intraflagellar transport (IFT), and cilia defects disrupt hedgehog signaling, causing many structural birth defects. We generated Ift25 and Ift27 knockout mice and show that they have structural birth defects indicative of hedgehog signaling dysfunction. Surprisingly, ciliary assembly is not affected, but abnormal hedgehog signaling is observed in conjunction with ciliary accumulation of patched-1 and Smoothened. Similarly, Smoothened accumulates in cilia on cells mutated for BBSome components or the BBS binding protein/regulator Lztfl1. Interestingly, the BBSome and Lztfl1 accumulate to high levels in Ift27 mutant cilia. Because Lztfl1 mutant cells accumulate BBSome but not IFT27, it is likely that Lztfl1 functions downstream of IFT27 to couple the BBSome to the IFT particle for coordinated removal of patched-1 and Smoothened from cilia during hedgehog signaling.

The catalytic subunit of the cAMP-dependent protein kinase of ovine sperm flagella has a unique amino-terminal sequence

The basis for the unusual properties of the catalytic subunit (C) of ram sperm cAMP-dependent protein kinase was investigated. Ram sperm C was purified and found by mass spectrometry (MS) to be ∼890 Da smaller than Cα, the predominant somatic isoform. Partial internal amino acid sequence from ram sperm C was an exact match to that of bovine Cα, but differed from the predicted sequences for the Cβ and Cγ isoforms. MS analysis of 2-nitro-5-thiocyanatobenzoic acid fragments showed that the mass difference originated in the amino-terminal region. A unique blocked amino-terminal fragment was isolated from sperm C and sequenced by a combination of tandem mass spectrometry and Edman degradation of a subfragment. The results revealed that the amino-terminal myristate and the first 14 amino acids of Cα are replaced by an amino-terminal acetate and six different amino acids in sperm C. The predicted mass difference due to these changes is 899 Da. The region of homology between sperm C and Cα begins at the exon 1/exon 2 boundary in Cα, suggesting that sperm C results from use of an alternate exon 1 in the Cα gene. The different amino terminus of sperm C may be related to a unique requirement for localization of the “free” C subunit within the sperm flagellum.

Wdpcp, a PCP Protein Required for Ciliogenesis, Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton

Wdpcp, a protein required for both planar cell polarity and ciliogenesis, regulates cell polarity and alignment via direct modulation of the actin cytoskeleton.

Differential expression of the C(s) and Calpha1 isoforms of the catalytic subunit of cyclic 3',5'-adenosine monophosphate-dependent protein kinase testicular cells

Abstract The amino terminus of the sperm cAMP-dependent protein kinase catalytic subunit (termed Cs) differs from that of the Cα1 isoform expressed in most tissues due to the use of alternative transcripts of the Cα gene. Both Cα1 and Cs transcripts are present in testis; Cs is expressed specifically in spermatogenic cells and is the only C isoform detected in mature sperm. Immunohistochemistry of mouse testis using antibodies specific for Cs and Cα1 now shows that Cα1 is present in somatic testicular cells, spermatogonia, and preleptotene spermatocytes but not in cells that are in later stages of spermatogenesis. In contrast, Cs is expressed only in midpachytene and later stage spermatocytes and in spermatids. Therefore, Cs and Cα1 expression do not overlap. Immunofluorescence microscopic localization of Cs in murine and ovine sperm reveals that Cs is located primarily in sperm tail components, including the midpiece mitochondria and the axoneme. Quantitative analysis of Western blots indicates that individual ovine sperm contain ∼4 × 105 molecules of Cs, a seemingly large number for a protein that acts catalytically.