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1.  BBS4 Is Necessary for Ciliary Localization of TrkB Receptor and Activation by BDNF 
PLoS ONE  2014;9(5):e98687.
Primary cilia regulate an expanding list of signaling pathways in many different cell types. It is likely that identification of the full catalog of pathways associated with cilia will be necessary to fully understand their role in regulation of signaling and the implications for diseases associated with their dysfunction, ciliopathies. Bardet-Biedl Syndrome (BBS) is one such ciliopathy which is characterized by a spectrum of phenotypes. These include neural defects such as impaired cognitive development, centrally mediated hyperphagia and peripheral sensory defects. Here we investigate potential defects in a signaling pathway associated with neuronal function, brain derived neurotrophic factor (BDNF) signaling. Upon loss of BBS4 expression in cultured cells, we observed decreased phosphorylation and activation by BDNF of its target receptor, TrkB. Assessment of ciliary localization revealed that, TrkB localized to the axonemes or basal bodies of cilia only in the presence of BDNF. Axonemal localization, specifically, was abrogated with loss of BBS4. Finally, we present evidence that loss of the ciliary axoneme through depletion of KIF3A impedes activation of TrkB. Taken together, these data suggest the possibility of a previously uninvestigated pathway associated with perturbation of ciliary proteins.
doi:10.1371/journal.pone.0098687
PMCID: PMC4035337  PMID: 24867303
2.  Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators 
The Journal of Clinical Investigation  2014;124(5):2059-2070.
Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients.
doi:10.1172/JCI71898
PMCID: PMC4001542  PMID: 24691443
3.  Functional modules, mutational load and human genetic disease 
Trends in genetics : TIG  2010;26(4):168-176.
The ability to generate a massive amount of sequencing and genotyping data is transforming the study of human genetic disorders. Driven by such innovation, it is likely that whole exome and whole-genome resequencing will replace regionally focused approaches for gene discovery and clinical testing in the next few years. However, this opportunity brings a significant interpretative challenge to assigning function and phenotypic variance to common and rare alleles. Understanding the effect of individual mutations in the context of the remaining genomic variation represents a major challenge to our interpretation of disease. Here, we discuss the challenges of assigning mutation functionality and, drawing from the examples of ciliopathies as well as cohesinopathies and channelopathies, discuss possibilities for the functional modularization of the human genome. Functional modularization in addition to the development of physiologically-relevant assays to test allele functionality will accelerate our understanding of disease architecture and enable the use of genome-wide sequence data for disease diagnosis and phenotypic prediction in individuals.
doi:10.1016/j.tig.2010.01.006
PMCID: PMC3740181  PMID: 20226561
4.  The emerging face of primary cilia 
Genesis (New York, N.Y. : 2000)  2011;49(4):231-246.
Primary cilia are microtubule-based organelles that serve as hubs for the transduction of various developmental signaling pathways including Hedgehog, Wnt, FGF and PDGF. Ciliary dysfunction contributes to a range of disorders, collectively known as the ciliopathies. Recently, interest has grown in these syndromes, particularly among craniofacial biologists, as many known and putative ciliopathies have severe craniofacial defects. Herein we discuss the current understanding of ciliary biology and craniofacial development in an attempt to gain insight into the molecular etiology for craniofacial ciliopathies, and uncover a characteristic ciliopathic craniofacial gestalt.
doi:10.1002/dvg.20728
PMCID: PMC3118297  PMID: 21305689
craniofacial; ciliopathy; hypertelorism; midline defects; Shh; Wnt
5.  Nde1-mediated inhibition of ciliogenesis affects cell cycle re-entry 
Nature cell biology  2011;13(4):351-360.
The primary cilium is an antenna-like organelle that is dynamically regulated during the cell cycle. Ciliogenesis is initiated as cells enter quiescence, while cilium resorption precedes mitosis. The mechanisms coordinating ciliogenesis with the cell cycle are unknown. Here we identify the centrosomal protein, Nde1, as a negative regulator of ciliary length. Nde1 is expressed at high levels in mitosis, low levels in quiescence and localizes at the mother centriole, which nucleates the primary cilium. Cells depleted of Nde1 show longer cilia and a delay in cell cycle re-entry that correlates with ciliary length. Knockdown of Nde1 in zebrafish embryos results in increased ciliary length, suppression of cell division, reduction of the number of cells forming the Kupffer’s vesicle, and left-right patterning defects. These data suggest that Nde1 is an integral component of a network coordinating ciliary length with cell cycle progression and have implications in the transition from quiescence to a proliferative state.
doi:10.1038/ncb2183
PMCID: PMC3077088  PMID: 21394081
6.  Mechanistic insights into Bardet-Biedl syndrome, a model ciliopathy 
Bardet-Biedl syndrome (BBS) is a multisystemic disorder typified by developmental and progressive degenerative defects. A combination of genetic, in vitro, and in vivo studies have highlighted ciliary dysfunction as a primary cause of BBS pathology, which has in turn contributed to the improved understanding of the functions of the primary cilium in humans and other vertebrates. Here we discuss the evidence linking the clinical BBS phenotype to ciliary defects, highlight how the genetic and cellular characteristics of BBS overlap with and inform other ciliary disorders, and explore the possible mechanistic underpinnings of ciliary dysfunction.
doi:10.1172/JCI37041
PMCID: PMC2648685  PMID: 19252258
7.  An Essential Role for DYF-11/MIP-T3 in Assembling Functional Intraflagellar Transport Complexes 
PLoS Genetics  2008;4(3):e1000044.
MIP-T3 is a human protein found previously to associate with microtubules and the kinesin-interacting neuronal protein DISC1 (Disrupted-in-Schizophrenia 1), but whose cellular function(s) remains unknown. Here we demonstrate that the C. elegans MIP-T3 ortholog DYF-11 is an intraflagellar transport (IFT) protein that plays a critical role in assembling functional kinesin motor-IFT particle complexes. We have cloned a loss of function dyf-11 mutant in which several key components of the IFT machinery, including Kinesin-II, as well as IFT subcomplex A and B proteins, fail to enter ciliary axonemes and/or mislocalize, resulting in compromised ciliary structures and sensory functions, and abnormal lipid accumulation. Analyses in different mutant backgrounds further suggest that DYF-11 functions as a novel component of IFT subcomplex B. Consistent with an evolutionarily conserved cilia-associated role, mammalian MIP-T3 localizes to basal bodies and cilia, and zebrafish mipt3 functions synergistically with the Bardet-Biedl syndrome protein Bbs4 to ensure proper gastrulation, a key cilium- and basal body-dependent developmental process. Our findings therefore implicate MIP-T3 in a previously unknown but critical role in cilium biogenesis and further highlight the emerging role of this organelle in vertebrate development.
Author Summary
The transport of protein complexes and associated cargo along microtubule tracks represents an essential eukaryotic process responsible for a multitude of cellular functions, including cell division, vesicle movement to membranes, and trafficking along dendrites, axons, and cilia. The latter organelles are hair-like cellular appendages implicated in cell and fluid motility, sensing and transducing information from their environment, and development. Their biogenesis and maintenance depends on a kinesin- and dynein-mediated motility process termed intraflagellar transport (IFT). In addition to comprising these specialized molecular motors, the IFT machinery consists of large multisubunit complexes whose exact composition and organization has not been fully defined. Here we identify a protein, DYF-11/MIP-T3, that is conserved in all ciliated organisms and is associated with IFT in C. elegans. Disruption of C. elegans DYF-11 results in structurally compromised cilia, likely as a result of IFT motor and subunit misassembly. Animals lacking DYF-11 display chemosensory anomalies, consistent with a role for the protein in cilia-associated sensory processes. In zebrafish, MIP-T3 is essential for gastrulation movements during development, similar to that observed for other ciliary components, including Bardet-Biedl syndrome proteins. In conclusion, we have identified a novel IFT machinery component that is also essential for development in vertebrates.
doi:10.1371/journal.pgen.1000044
PMCID: PMC2268012  PMID: 18369462
8.  Changes in Rx1 and Pax6 activity at eye field stages differentially alter the production of amacrine neurotransmitter subtypes in Xenopus 
Molecular Vision  2007;13:86-95.
Purpose
Both rx1 and pax6 are expressed during the initial formation of the vertebrate eye field, and they are thought to be crucial for maintenance of the retinal stem cells in the ciliary marginal zone. However, both genes continue to be expressed in different layers of the differentiating retina, suggesting that they have additional roles in cell type specification. Because previous work suggested that amacrine cell subtypes are derived from biased progenitors in the eye field, we tested whether altering Rx1 or Pax6 activity during eye field stages affects the production of three neurotransmitter subtypes of amacrine cells.
Methods
Gain-of-function and loss-of-function hormone-inducible constructs of Rx1 and Pax6 were used to alter Rx1 and Pax6 protein or activity levels after the formation of the eye field. The major-retina producing blastomere of the 32-cell stage Xenopus embryo (D1.1.1) was injected with mRNA encoding one of these proteins and mRNA encoding GFP to label the altered lineage. Embryos were treated with synthetic hormone at either early (stage 12) or late (stage 16) eye field stages and they developed to tadpole stages (stage 44/45) when the cells in the central retina have differentiated. Amacrine cell subtypes (dopamine [DA], neuropeptide Y [NPY], γ aminobutyrate acid [GABA]) were detected by immunofluorescence histology and the numbers of each type of cell produced within the affected lineage were counted. The percent contribution of the D1.1.1 lineage to a particular amacrine subtype after stage 12 or stage 16 hormone treatment were independently compared to those from gfp mRNA-injected control embryos that were similarly treated with hormone.
Results
Increasing Rx1 at early eye field stages promotes NPY amacrine cells and represses GABA and DA amacrine cells, and at late eye field stages significantly represses DA and NPY phenotypes but has a diminished effect on the GABA phenotype. Increasing Pax6 at early eye field stages represses NPY and DA amacrine cells but does not affect the GABA phenotype, whereas in the late eye field it significantly represses only the DA phenotype.
Conclusions
Rx1 and Pax6 differentially modify the ability of eye field precursors to produce different neurotransmitter subtypes of amacrine cells. These effects varied for each of the subtypes investigated, indicating that amacrine cells are not all specified by a single genetic program. Furthermore, some cases were time-dependent, indicating that the downstream effects change as development proceeds.
PMCID: PMC2503186  PMID: 17277735

Results 1-8 (8)