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1.  Fuz Regulates Craniofacial Development through Tissue Specific Responses to Signaling Factors 
PLoS ONE  2011;6(9):e24608.
The planar cell polarity effector gene Fuz regulates ciliogenesis and Fuz loss of function studies reveal an array of embryonic phenotypes. However, cilia defects can affect many signaling pathways and, in humans, cilia defects underlie several craniofacial anomalies. To address this, we analyzed the craniofacial phenotype and signaling responses of the Fuz−/− mice. We demonstrate a unique role for Fuz in regulating both Hedgehog (Hh) and Wnt/β-catenin signaling during craniofacial development. Fuz expression first appears in the dorsal tissues and later in ventral tissues and craniofacial regions during embryonic development coincident with cilia development. The Fuz−/− mice exhibit severe craniofacial deformities including anophthalmia, agenesis of the tongue and incisors, a hypoplastic mandible, cleft palate, ossification/skeletal defects and hyperplastic malformed Meckel's cartilage. Hh signaling is down-regulated in the Fuz null mice, while canonical Wnt signaling is up-regulated revealing the antagonistic relationship of these two pathways. Meckel's cartilage is expanded in the Fuz−/− mice due to increased cell proliferation associated with the up-regulation of Wnt canonical target genes and decreased non-canonical pathway genes. Interestingly, cilia development was decreased in the mandible mesenchyme of Fuz null mice, suggesting that cilia may antagonize Wnt signaling in this tissue. Furthermore, expression of Fuz decreased expression of Wnt pathway genes as well as a Wnt-dependent reporter. Finally, chromatin IP experiments demonstrate that β-catenin/TCF-binding directly regulates Fuz expression. These data demonstrate a new model for coordination of Hh and Wnt signaling and reveal a Fuz-dependent negative feedback loop controlling Wnt/β-catenin signaling.
doi:10.1371/journal.pone.0024608
PMCID: PMC3173472  PMID: 21935430
2.  Control of vertebrate intraflagellar transport by the planar cell polarity effector Fuz 
The Journal of Cell Biology  2012;198(1):37-45.
The planar cell polarity effector Fuz is required for normal particle dynamics of the intraflagellar transport system, specifically in the retrograde transport of proteins.
Cilia play key roles in development and homeostasis, and defects in cilia structure or function lead to an array of human diseases. Ciliogenesis is accomplished by the intraflagellar transport (IFT) system, a set of proteins governing bidirectional transport of cargoes within ciliary axonemes. In this paper, we present a novel platform for in vivo analysis of vertebrate IFT dynamics. Using this platform, we show that the planar cell polarity (PCP) effector Fuz was required for normal IFT dynamics in vertebrate cilia, the first evidence directly linking PCP to the core machinery of ciliogenesis. Further, we show that Fuz played a specific role in trafficking of retrograde, but not anterograde, IFT proteins. These data place Fuz in the small group of known IFT effectors outside the core machinery and, additionally, identify Fuz as a novel cytoplasmic effector that differentiates between the retrograde and anterograde IFT complexes.
doi:10.1083/jcb.201204072
PMCID: PMC3392940  PMID: 22778277
3.  Wdpcp, a PCP Protein Required for Ciliogenesis, Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton 
PLoS Biology  2013;11(11):e1001720.
Wdpcp, a protein required for both planar cell polarity and ciliogenesis, regulates cell polarity and alignment via direct modulation of the actin cytoskeleton.
Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet–Biedl/Meckel–Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration.
Author Summary
Cilia are microscopic cell surface hair-like protrusions that can act as antennae to mediate cell signaling. Mutations disrupting ciliogenesis can cause many developmental anomalies associated with syndromes known as “ciliopathies.” Some developmental defects, such as limb polydactyly, arise from disruption of cilia-transduced sonic hedgehog signaling, while other defects, such as aberrant patterning of hair cells in the inner ear, arise from disrupted Wnt signaling resulting in modulation of planar cell polarity (PCP)—a process whereby cells are polarized and aligned. While ciliopathy phenotypes would suggest that cilia are involved in modulating PCP, the mechanistic link between cilia and PCP has been elusive. Our study using a mouse model carrying a mutation in Wdpcp, a gene required for both ciliogenesis and PCP, suggest that Wdpcp modulation of PCP involves interactions with the actin cytoskeleton separate from its function in ciliogenesis. We observe Wdpcp localization in cilia, where it is required for recruitment of proteins essential for ciliogenesis. Wdpcp interacts with Sept2, and is also found in actin filaments, where it regulates actin dynamics essential for PCP. Together, these findings show that PCP regulation by Wdpcp is distinct from its function in ciliogenesis and involves direct modulation of the actin cytoskeleton.
doi:10.1371/journal.pbio.1001720
PMCID: PMC3841097  PMID: 24302887
4.  The PCP effector Fuzzy controls cilial assembly and signaling by recruiting Rab8 and Dishevelled to the primary cilium 
Molecular Biology of the Cell  2013;24(5):555-565.
During vertebrate development, the PCP pathway controls multiple cellular processes. Loss of the gene for the PCP effector Fuzzy affects formation of primary cilia via mostly unknown mechanisms. We report that Fuzzy localizes to the primary cilia and orchestrates delivery of Rab8 and Dishevelled to the primary cilium; loss of Fuzzy affects cilia-dependent signaling.
The planar cell polarity (PCP) pathway controls multiple cellular processes during vertebrate development. Recently the PCP pathway was implicated in ciliogenesis and in ciliary function. The primary cilium is an apically projecting solitary organelle that is generated via polarized intracellular trafficking. Because it acts as a signaling nexus, defects in ciliogenesis or cilial function cause multiple congenital anomalies in vertebrates. Loss of the PCP effector Fuzzy affects PCP signaling and formation of primary cilia; however, the mechanisms underlying these processes are largely unknown. Here we report that Fuzzy localizes to the basal body and ciliary axoneme and is essential for ciliogenesis by delivering Rab8 to the basal body and primary cilium. Fuzzy appears to control subcellular localization of the core PCP protein Dishevelled, recruiting it to Rab8-positive vesicles and to the basal body and cilium. We show that loss of Fuzzy results in inhibition of PCP signaling and hyperactivation of the canonical WNT pathway. We propose a mechanism by which Fuzzy participates in ciliogenesis and affects both canonical WNT and PCP signaling.
doi:10.1091/mbc.E12-06-0437
PMCID: PMC3583660  PMID: 23303251
5.  The Small GTPase Rsg1 is important for the cytoplasmic localization and axonemal dynamics of intraflagellar transport proteins 
Cilia  2013;2:13.
Background
Cilia are small, microtubule-based protrusions important for development and homeostasis. We recently demonstrated that the planar cell polarity effector protein Fuz is a critical regulator of axonemal intraflagellar transport dynamics and localization. Here, we report our findings on the role of the small GTPase Rsg1, a known binding partner of Fuz, and its role in the dynamics and cytoplasmic localization of intraflagellar transport proteins.
Results
We find that Rsg1 loss of function leads to impaired axonemal IFT dynamics in multiciliated cells. We further show that Rsg1 is required for appropriate cytoplasmic localization of the retrograde IFT-A protein IFT43. Finally, we show that Rsg1 governs the apical localization of basal bodies, the anchoring structures of cilia.
Conclusions
Our data suggest that Rsg1 is a regulator of multiple aspects of ciliogenesis, including apical trafficking of basal bodies and the localization and dynamics intraflagellar transport proteins.
doi:10.1186/2046-2530-2-13
PMCID: PMC3850895  PMID: 24192041
Cilia; Fuz; IFT; PCP; Rsg1
6.  Planar cell polarity effector gene Intu regulates cell fate-specific differentiation of keratinocytes through the primary cilia 
Cell Death and Differentiation  2012;20(1):130-138.
Genes involved in the planar cell polarity (PCP) signaling pathway are essential for a number of developmental processes in mammals, such as convergent extension and ciliogenesis. Tissue-specific PCP effector genes of the PCP signaling pathway are believed to mediate PCP signals in a tissue- and cell type-specific manner. However, how PCP signaling controls the morphogenesis of mammalian tissues remains unclear. In this study, we investigated the role of inturned (Intu), a tissue-specific PCP effector gene, during hair follicle formation in mice. Tissue-specific disruption of Intu in embryonic epidermis resulted in hair follicle morphogenesis arrest because of the failure of follicular keratinocyte to differentiate. Targeting Intu in the epidermis resulted in almost complete loss of primary cilia in epidermal and follicular keratinocytes, and a suppressed hedgehog signaling pathway. Surprisingly, the epidermal stratification and differentiation programs and barrier function were not affected. These results demonstrate that tissue-specific PCP effector genes of the PCP signaling pathway control the differentiation of keratinocytes through the primary cilia in a cell fate- and context-dependent manner, which may be critical in orchestrating the propagation and interpretation of polarity signals established by the core PCP components.
doi:10.1038/cdd.2012.104
PMCID: PMC3524640  PMID: 22935613
Planar cell polarity; Intu; cilia; keratinocyte; epidermis; hair follicle
7.  Genome-Wide RNAi Screen Identifies Novel Host Proteins Required for Alphavirus Entry 
PLoS Pathogens  2013;9(12):e1003835.
The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ), a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.
Author Summary
Alphaviruses are a group of small enveloped viruses that include important human pathogens for which there are no antiviral therapies or vaccines. Alphaviruses enter host cells by receptor-mediated endocytosis and low pH-triggered membrane fusion, and exit by budding from the host cell plasma membrane. The roles of host cell proteins in these events are not well understood in spite of extensive studies. Here we performed a screen using small interfering RNAs to identify host factors involved in alphavirus infection of human cells. We defined the mechanism of two novel host proteins that promote alphavirus entry. Fuzzy homologue (FUZ), a protein with roles in cilia biogenesis, promoted endocytosis of both alphaviruses and a well-studied endocytic cargo, transferrin. The tetraspanin membrane protein, TSPAN9, did not significantly affect endocytic uptake or acidification, but was critical for the efficient fusion of the virus in the endosome. These two proteins were required for infection by several different alphaviruses, suggesting that they may be useful targets for drugs to prevent alphavirus infection.
doi:10.1371/journal.ppat.1003835
PMCID: PMC3868536  PMID: 24367265
8.  A hypomorphic allele reveals an important role of Inturned in mouse skeletal development 
Background
Cilia are important for Hedgehog signaling in vertebrates and many genes that encode proteins involved in ciliogenesis have been studied for their roles in embryonic development. Null mutations in many of these genes cause early embryonic lethality, hence an understanding of their roles in postnatal development is limited.
Results
The Inturned (Intu) gene is required for ciliogenesis and here we report a recessive hypomorphic mutation, resulting in substitution of a conserved hydrophobic residue (I813N) near the C-terminus, that sheds light on later functions of Intu. Mice homozygous for this Double-thumb (IntuDtm) allele exhibit polydactyly, retarded growth, and reduced survival. There is a moderate loss of cilia in IntuDtm/Dtm mutants, and IntuI813N exhibits compromised ability to increase ciliogenesis in cultured Intu null mutant cells. IntuDtm mutants show rib defects and delay of endochondral ossification in long bones, digits, vertebrae and the sternum. These skeletal defects correlate with a decrease in Hh signaling. However, patterning of the neural tube and planar cell polarity appear to be normal.
Conclusion
This hypomorphic Intu allele highlights an important role of Intu in mouse skeletal development.
doi:10.1002/dvdy.24272
PMCID: PMC4449324  PMID: 25774014
Hedgehog signaling; polydactyly; primary cilia; ciliogenesis; skeletal development; endochondral ossification
9.  Mating and Pathogenic Development of the Smut Fungus Ustilago maydis Are Regulated by One Mitogen-Activated Protein Kinase Cascade 
Eukaryotic Cell  2003;2(6):1187-1199.
In the phytopathogenic fungus Ustilago maydis, pheromone-mediated cell fusion is a prerequisite for the generation of the infectious dikaryon. The pheromone signal elevates transcription of the pheromone genes and elicits formation of conjugation hyphae. Cyclic AMP and mitogen-activated protein kinase (MAPK) signaling are involved in this process. The MAPK cascade is presumed to be composed of Ubc4 (MAPK kinase kinase), Fuz7 (MAPK kinase), and Ubc3/Kpp2 (MAPK). We isolated the kpp4 gene and found it to be allelic to ubc4. Epistasis analyses with constitutively active alleles of kpp4 and fuz7 substantiate that Kpp4, Fuz7, and Kpp2/Ubc3 are components of the same module. Moreover, we demonstrate that Fuz7 activates Kpp2 and shows interactions in vitro. Signaling via this cascade regulates expression of pheromone-responsive genes, presumably through acting on the transcription factor Prf1. Interestingly, the same cascade is needed for conjugation tube formation, and this process does not involve Prf1. In addition, fuz7 as well as kpp4 deletion strains are nonpathogenic, while kpp2 deletion mutants are only attenuated in pathogenesis. Here we show that strains expressing the unphosphorylatable allele kpp2T182A/Y184F are severely affected in tumor induction and display defects in early infection-related differentiation.
doi:10.1128/EC.2.6.1187-1199.2003
PMCID: PMC326639  PMID: 14665454
10.  Microtubules Enable the Planar Cell Polarity of Airway Cilia 
Current biology : CB  2012;22(23):2203-2212.
Summary
Background
Airway cilia must be physically oriented along the longitudinal tissue axis for concerted, directional motility that is essential for proper mucociliary clearance.
Results
We show that Planar Cell Polarity (PCP) signaling specifies directionality and orients respiratory cilia. Within all airway epithelial cells a conserved set of PCP proteins shows interdependent, asymmetric junctional localization; non-autonomous signaling coordinates polarization between cells; and a polarized microtubule (MT) network is likely required for asymmetric PCP protein localization. We find that basal bodies dock after polarity of PCP proteins is established, are polarized nearly simultaneously, and refinement of basal body/cilium orientation continues during airway epithelial development. Unique to mature multiciliated cells, we identify PCP-regulated, planar polarized MTs that originate from basal bodies and interact, via their plus ends, with membrane domains associated with the PCP proteins Frizzled and Dishevelled. Disruption of MTs leads to misoriented cilia.
Conclusions
A conserved PCP pathway orients airway cilia by communicating polarity information from asymmetric membrane domains at the apical junctions, through MTs, to orient the MT and actin based network of ciliary basal bodies below the apical surface.
doi:10.1016/j.cub.2012.09.046
PMCID: PMC3518597  PMID: 23122850
11.  Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation 
JCI Insight  null;1(13):e88027.
Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease.
Planar cell polarity signaling regulates mucociliary clearance, barrier function, and regeneration in airway epithelia, and depends on the acquisition of multiciliated cell fate.
doi:10.1172/jci.insight.88027
PMCID: PMC4996276  PMID: 27570836
12.  Flattop regulates basal body docking and positioning in mono- and multiciliated cells 
eLife  2014;3:e03842.
Planar cell polarity (PCP) regulates basal body (BB) docking and positioning during cilia formation, but the underlying mechanisms remain elusive. In this study, we investigate the uncharacterized gene Flattop (Fltp) that is transcriptionally activated during PCP acquisition in ciliated tissues. Fltp knock-out mice show BB docking and ciliogenesis defects in multiciliated lung cells. Furthermore, Fltp is necessary for kinocilium positioning in monociliated inner ear hair cells. In these cells, the core PCP molecule Dishevelled 2, the BB/spindle positioning protein Dlg3, and Fltp localize directly adjacent to the apical plasma membrane, physically interact and surround the BB at the interface of the microtubule and actin cytoskeleton. Dlg3 and Fltp knock-outs suggest that both cooperatively translate PCP cues for BB positioning in the inner ear. Taken together, the identification of novel BB/spindle positioning components as potential mediators of PCP signaling might have broader implications for other cell types, ciliary disease, and asymmetric cell division.
DOI: http://dx.doi.org/10.7554/eLife.03842.001
eLife digest
Epithelial tissues are sheets of cells that line the surface of many parts of the body, including the airways and the inner ear. Small hair-like structures called cilia can be found on the top surface of many epithelial cells and are arranged in a precise, ordered pattern. Such patterning ensures that cilia can work in a co-ordinated manner, for example by beating together to help clearing mucus from airways.
Cilia grow out from ‘basal bodies’ and, like many other important structures in a cell, these basal bodies must be oriented along the correct side of an epithelial tissue. This is achieved by ‘planar cell polarity signaling’, which makes sure that the structures inside a cell are correctly aligned, and ensures that polarized cells themselves are correctly oriented across the epithelial tissue. Disruption of this signaling can result in developmental defects.
Some proteins help to establish polarity in a cell by altering the cell's cytoskeleton—the structural support and transport network of the cell. A ‘core’ complex of proteins then coordinates how the cells are arranged throughout the epithelial tissue. Although many of the proteins involved in each of these roles are known, how they interact with each other to establish planar cell polarity remains poorly understood.
Now, Gegg et al. report that, in mice, a protein called Flattop functions to position basal bodies—and thus cilia—by working together with another protein called Dlg3. In mice that cannot produce Flattop, cilia formation is defective in the lung, and the cilia in the inner ear are positioned incorrectly. Gegg et al. found that in the inner ear, Flattop and Dlg3 physically interact with each other and two other proteins—including one of the core proteins involved in planar cell polarity. This protein complex then surrounds the basal bodies at the point where they connect to the cell's cytoskeleton.
Future challenges will be to clarify how the protein complex anchors to the cytoskeleton and how it interacts with other core planar cell polarity proteins in the cells of the inner ear. It will also be important to see whether this protein complex fulfills a similar role in other ciliated epithelial tissues.
DOI: http://dx.doi.org/10.7554/eLife.03842.002
doi:10.7554/eLife.03842
PMCID: PMC4221739  PMID: 25296022
basal body; spindle positioning; planar cell polarity; actin; microtubule; Flattop; mouse
13.  Drosophila CK1-γ, gilgamesh, controls PCP-mediated morphogenesis through regulation of vesicle trafficking 
The Journal of Cell Biology  2012;196(5):605-621.
CK1-γ/gilgamesh spatially limits the planar cell polarity–regulated process of trichome formation in Drosophila through its effect on polarized vesicle recycling.
Cellular morphogenesis, including polarized outgrowth, promotes tissue shape and function. Polarized vesicle trafficking has emerged as a fundamental mechanism by which protein and membrane can be targeted to discrete subcellular domains to promote localized protrusions. Frizzled (Fz)/planar cell polarity (PCP) signaling orchestrates cytoskeletal polarization and drives morphogenetic changes in such contexts as the vertebrate body axis and external Drosophila melanogaster tissues. Although regulation of Fz/PCP signaling via vesicle trafficking has been identified, the interplay between the vesicle trafficking machinery and downstream terminal PCP-directed processes is less established. In this paper, we show that Drosophila CK1-γ/gilgamesh (gish) regulates the PCP-associated process of trichome formation through effects on Rab11-mediated vesicle recycling. Although the core Fz/PCP proteins dictate prehair formation broadly, CK1-γ/gish restricts nucleation to a single site. Moreover, CK1-γ/gish works in parallel with the Fz/PCP effector multiple wing hairs, which restricts prehair formation along the perpendicular axis to Gish. Our findings suggest that polarized Rab11-mediated vesicle trafficking regulated by CK1-γ is required for PCP-directed processes.
doi:10.1083/jcb.201107137
PMCID: PMC3307696  PMID: 22391037
14.  Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation 
JCI insight  2016;1(13):e88027.
Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease.
doi:10.1172/jci.insight.88027
PMCID: PMC4996276  PMID: 27570836
15.  Fuz1, a MYND domain protein, is required for cell morphogenesis in Ustilago maydis 
Mycologia  2008;100(1):31-46.
Ustilago maydis is a Basidiomycete fungus that exhibits a yeast-like nonpathogenic form and a dikaryotic filamentous pathogenic form. Generation of these two forms is controlled by two mating type loci, a and b. The fungus undergoes additional morphological transitions in the plant that result in formation of a third cell type, the teliospore. The fuz1 gene is necessary for this developmental program. Here we report cloning and sequencing of fuz1 and show that it contains an open reading frame with coding capacity for a protein of 1421 amino acids. The Fuz1 protein belongs to the family of MYND Zn finger domain proteins. We generate a null mutation in strains of opposite mating type and show that fuz1 is necessary for conjugation tube formation, a morphological transition that occurs in response to pheromones. We generate fuz1− diploid strains heterozygous at a and b and show that fuz1 is also necessary for postfusion events (maintenance of filamentous growth). We also demonstrate that fuz1 is necessary for cell morphogenesis of the yeast-like cell: normal cell length, location and number of septa, cell separation and constriction of the neck region. Fuz1 is also required for cell wall integrity and to prevent secretion of a dark pigment. We propose that the MYND domain may interact with different proteins to regulate cell morphogenesis.
PMCID: PMC2556375  PMID: 18488351
cell separation; conjugation tube; filamentous growth; pigment production; septum location
16.  Antagonistic Functions of Dishevelleds Regulate Frizzled3 Endocytosis via Filopodia Tips in Wnt-Mediated Growth Cone Guidance 
The Journal of Neuroscience  2013;33(49):19071-19085.
How growth cones detect small concentration differences of guidance cues for correct steering remains a long-standing puzzle. Commissural axons engage planar cell polarity (PCP) signaling components to turn anteriorly in a Wnt gradient after midline crossing. We found here that Frizzled3, a Wnt receptor, undergoes endocytosis via filopodia tips. Wnt5a increases Frizzled3 endocytosis, which correlates with filopodia elongation. We discovered an unexpected antagonism between Dishevelleds, which may function as a signal amplification mechanism in filopodia where PCP signaling is activated: Dishevelled2 blocks Dishevelled1-induced Frizzled3 hyperphosphorylation and membrane accumulation. A key component of apical-basal polarity (A-BP) signaling, aPKC, also inhibits Dishevelled1-induced Frizzled3 hyperphosphorylation. Celsr3, another PCP component, is required in commissural neurons for anterior turning. Frizzled3 hyperphosphorylation is increased in Celsr3 mutant mice, where PCP signaling is impaired, suggesting Frizzled3 hyperphosphorylation does correlate with loss of PCP signaling in vivo. Furthermore, we found that the small GTPase, Arf6, which is required for Frizzled3 endocytosis, is essential for Wnt-promoted outgrowth, highlighting the importance of Frizzled3 recycling in PCP signaling in growth cone guidance. In a Wnt5a gradient, more Frizzled3 endocytosis and activation of atypical protein kinase C was observed on the side of growth cones facing higher Wnt5a concentration, suggesting that spatially controlled Frizzled3 endocytosis is part of the key mechanism for growth cone steering.
doi:10.1523/JNEUROSCI.2800-13.2013
PMCID: PMC3850035  PMID: 24305805
17.  The involvement of PCP proteins in radial cell intercalations during Xenopus embryonic development 
Developmental biology  2015;408(2):316-327.
The planar cell polarity (PCP) pathway orients cells in diverse epithelial tissues in Drosophila and vertebrate embryos and has been implicated in many human congenital defects and diseases, such as ciliopathies, polycystic kidney disease and malignant cancers. During vertebrate gastrulation and neurulation, PCP signaling is required for convergent extension movements, which are primarily driven by mediolateral cell intercalations, whereas the role for PCP signaling in radial cell intercalations has been unclear. In this study, we examine the function of the core PCP proteins Vangl2, Prickle3 (Pk3) and Disheveled in the ectodermal cells, which undergo radial intercalations during Xenopus gastrulation and neurulation. In the epidermis, multiciliated cell (MCC) progenitors originate in the inner layer, but subsequently migrate to the embryo surface during neurulation. We find that the Vangl2/Pk protein complexes are enriched at the apical domain of intercalating MCCs and are essential for the MCC intercalatory behavior. Addressing the underlying mechanism, we identified KIF13B, as a motor protein that binds Disheveled. KIF13B is required for MCC intercalation and acts synergistically with Vangl2 and Disheveled, indicating that it may mediate microtubule-dependent trafficking of PCP proteins necessary for cell shape regulation. In the neural plate, the Vangl2/Pk complexes were also concentrated near the outermost surface of deep layer cells, suggesting a general role for PCP in radial intercalation. Consistent with this hypothesis, the ectodermal tissues deficient in Vangl2 or Disheveled functions contained more cell layers than normal tissues. We propose that PCP signaling is essential for both mediolateral and radial cell intercalations during vertebrate morphogenesis. These expanded roles underscore the significance of vertebrate PCP proteins as factors contributing to a number of diseases, including neural tube defects, tumor metastases, and various genetic syndromes characterized by abnormal migratory cell behaviors.
doi:10.1016/j.ydbio.2015.06.013
PMCID: PMC4810801  PMID: 26079437
Planar cell polarity; Radial intercalation; Multiciliated cells; Vangl2; Prickle3; Disheveled; KIF13B; Xenopus; Gastrulation
18.  The Drosophila Homologue of the Amyloid Precursor Protein Is a Conserved Modulator of Wnt PCP Signaling 
PLoS Biology  2013;11(5):e1001562.
The Drosophila homolog of the Alzheimer's disease protein APP, known as APPL, regulates axon growth during brain development.
Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.
Author Summary
Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but in neurons it regulates axon outgrowth, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid Precursor Proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. In the present work we investigate the role of the Drosophila neuron-specific APP homologue, called APPL, during brain development. We find that APPL is required for the development of αβ neurons in the mushroom body, a structure critical for learning and memory. We find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not for cell polarity. Molecularly, both human APP and fly APPL are found in membrane complexes with PCP receptors. Moreover, we show that APPL regulates PCP pathway activation through its downstream effector Abelson kinase (Abl), which modulates the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) and the subsequent activation of Wnt-PCP signaling. Taken together our data suggest that APPL is the first example of a neuron-specific modulator of the Wnt-PCP pathway.
doi:10.1371/journal.pbio.1001562
PMCID: PMC3653798  PMID: 23690751
19.  Mechanosensory Genes Pkd1 and Pkd2 Contribute to the Planar Polarization of Brain Ventricular Epithelium 
The Journal of Neuroscience  2015;35(31):11153-11168.
Directional beating of ependymal (E) cells' cilia in the walls of the ventricles in the brain is essential for proper CSF flow. E cells display two forms of planar cell polarity (PCP): rotational polarity of individual cilium and translational polarity (asymmetric positioning of cilia in the apical area). The orientation of individual E cells varies according to their location in the ventricular wall (location-specific PCP). It has been hypothesized that hydrodynamic forces on the apical surface of radial glia cells (RGCs), the embryonic precursors of E cells, could guide location-specific PCP in the ventricular epithelium. However, the detection mechanisms for these hydrodynamic forces have not been identified. Here, we show that the mechanosensory proteins polycystic kidney disease 1 (Pkd1) and Pkd2 are present in primary cilia of RGCs. Ablation of Pkd1 or Pkd2 in Nestin-Cre;Pkd1flox/flox or Nestin-Cre;Pkd2flox/flox mice, affected PCP development in RGCs and E cells. Early shear forces on the ventricular epithelium may activate Pkd1 and Pkd2 in primary cilia of RGCs to properly polarize RGCs and E cells. Consistently, Pkd1, Pkd2, or primary cilia on RGCs were required for the proper asymmetric localization of the PCP protein Vangl2 in E cells' apical area. Analyses of single- and double-heterozygous mutants for Pkd1 and/or Vangl2 suggest that these genes function in the same pathway to establish E cells' PCP. We conclude that Pkd1 and Pkd2 mechanosensory proteins contribute to the development of brain PCP and prevention of hydrocephalus.
SIGNIFICANCE STATEMENT This study identifies key molecules in the development of planar cell polarity (PCP) in the brain and prevention of hydrocephalus. Multiciliated ependymal (E) cells within the brain ventricular epithelium generate CSF flow through ciliary beating. E cells display location-specific PCP in the orientation and asymmetric positioning of their cilia. Defects in this PCP can result in hydrocephalus. Hydrodynamic forces on radial glial cells (RGCs), the embryonic progenitors of E cells, have been suggested to guide PCP. We show that the mechanosensory proteins Pkd1 and Pkd2 localize to primary cilia in RGCs, and their ablation disrupts the development of PCP in E cells. Early shear forces on RGCs may activate Pkd1 and Pkd2 in RGCs' primary cilia to properly orient E cells. This study identifies key molecules in the development of brain PCP and prevention of hydrocephalus.
doi:10.1523/JNEUROSCI.0686-15.2015
PMCID: PMC4524982  PMID: 26245976
cilia; ependymal cell; epithelium; neural stem cell; planar cell polarity; polycystin
20.  Casein kinase 1δ functions at the centrosome and Golgi to promote ciliogenesis 
Molecular Biology of the Cell  2014;25(10):1629-1640.
CK1δ acts at the centrosome and Golgi to support polarized transport for ciliogenesis. It controls distribution of ciliary effectors Rab11, Rab8, CEP290, PCM1, and IFT20 and also promotes MT nucleation at the Golgi and positioning and integrity of the Golgi. Interaction of CK1δ with AKAP450 mediates Golgi MT nucleation and ciliogenesis.
Inhibition of casein kinase 1 delta (CK1δ) blocks primary ciliogenesis in human telomerase reverse transcriptase immortalized retinal pigmented epithelial and mouse inner medullary collecting duct cells-3. Mouse embryonic fibroblasts (MEFs) and retinal cells from Csnk1d (CK1δ)-null mice also exhibit ciliogenesis defects. CK1δ catalytic activity and centrosomal localization signal (CLS) are required to rescue cilia formation in MEFsCsnk1d null. Furthermore, expression of a truncated derivative containing the CLS displaces full-length CK1δ from the centrosome and decreases ciliary length in control MEFs, suggesting that centrosomal CK1δ has a role in ciliogenesis. CK1δ inhibition also alters pericentrosomal or ciliary distribution of several proteins involved in ciliary transport, including Ras-like in rat brain-11A, Ras-like in rat brain-8A, centrosomal protein of 290 kDa, pericentriolar material protein 1, and polycystin-2, as well as the Golgi distribution of its binding partner, A-kinase anchor protein 450 (AKAP450). As reported for AKAP450, CK1δ was required for microtubule nucleation at the Golgi and maintenance of Golgi integrity. Overexpression of an AKAP450 fragment containing the CK1δ-binding site inhibits Golgi-derived microtubule nucleation, Golgi distribution of intraflagellar transport protein 20 homologue, and ciliogenesis. Our results suggest that CK1δ mediates primary ciliogenesis by multiple mechanisms, one involving its centrosomal function and another dependent on its interaction with AKAP450 at the Golgi, where it is important for maintaining Golgi organization and polarized trafficking of multiple factors that mediate ciliary transport.
doi:10.1091/mbc.E13-10-0598
PMCID: PMC4019494  PMID: 24648492
21.  Frizzled-induced Van Gogh phosphorylation by CK1ε promotes asymmetric localization of core PCP factors in Drosophila 
Cell reports  2016;16(2):344-356.
Epithelial tissues are polarized along two axes. In addition to apical-basal polarity they are often polarized within the plane of the epithelium, so-called Planar Cell Polarity (PCP). PCP depends upon Wnt/Frizzled (Fz) signaling factors, including Fz itself and Van Gogh (Vang/Vangl). We sought to understand how Vang interaction with other core PCP factors affects Vang function. We find that Fz induces Vang phosphorylation in a cell-autonomous manner. Vang phosphorylation occurs on conserved N-terminal serine/threonine residues, is mediated by CK1ε/Dco and is critical for polarized membrane localization of Vang and other PCP proteins. This regulatory mechanism does not require Fz signaling through Dishevelled, and thus represents a cell-autonomous upstream interaction between Fz and Vang. Furthermore, this signaling event appears to be related to Wnt5a-mediated Vangl2 phosphorylation during mouse limb patterning, and may thus be a general mechanism underlying Wnt-regulated PCP establishment.
Graphical Abstract
eTOC blurb
Kelly et al. find that Vang phosphorylation is involved in Planar Cell Polarity (PCP) signaling. This phosphorylation is mediated by CK1ε/Dco and requires cell-autonomous Frizzled signaling activity, but is independent of Dsh function. Vang phosphorylation is essential for polarized membrane localization and Vang function in PCP.
doi:10.1016/j.celrep.2016.06.010
PMCID: PMC4945453  PMID: 27346358
22.  Serrano (Sano) Functions with the Planar Cell Polarity Genes to Control Tracheal Tube Length 
PLoS Genetics  2009;5(11):e1000746.
Epithelial tubes are the functional units of many organs, and proper tube geometry is crucial for organ function. Here, we characterize serrano (sano), a novel cytoplasmic protein that is apically enriched in several tube-forming epithelia in Drosophila, including the tracheal system. Loss of sano results in elongated tracheae, whereas Sano overexpression causes shortened tracheae with reduced apical boundaries. Sano overexpression during larval and pupal stages causes planar cell polarity (PCP) defects in several adult tissues. In Sano-overexpressing pupal wing cells, core PCP proteins are mislocalized and prehairs are misoriented; sano loss or overexpression in the eye disrupts ommatidial polarity and rotation. Importantly, Sano binds the PCP regulator Dishevelled (Dsh), and loss or ectopic expression of many known PCP proteins in the trachea gives rise to similar defects observed with loss or gain of sano, revealing a previously unrecognized role for PCP pathway components in tube size control.
Author Summary
Tubular organ formation is a ubiquitous process required to sustain life in multicellular organisms. In this study, we focused on the tracheal system of the fruit fly, Drosophila melanogaster, and identified Serrano (Sano) as a novel protein expressed in several embryonic tubular organs, including trachea. sano loss results in over-elongated trachea, whereas Sano overexpression causes shortened trachea, suggesting that sano is required for proper tracheal tube length. Interestingly, Sano overexpression results in typical planar cell polarity (PCP) defects in many adult tissues and pupal wing cells. The PCP pathway is highly conserved from flies to mammals and it has been known to control cell polarity within the plane of epithelial tissues. Importantly, we found that Sano binds Dishevelled (Dsh), a key PCP regulator, and loss or ectopic expression of many known PCP proteins in the trachea give rise to similar defects observed with loss or gain of sano, suggesting a new role for the PCP genes in tube length control. Interestingly, the changes in tube length and PCP defects in the wing were linked to changes in apical domain size, suggesting that Sano and the PCP components affect either membrane recycling and/or the linkage of the membrane to the cytoskeleton.
doi:10.1371/journal.pgen.1000746
PMCID: PMC2776533  PMID: 19956736
23.  Dishevelled controls apical docking and planar polarization of basal bodies in ciliated epithelial cells 
Nature genetics  2008;40(7):871-879.
The planar cell polarity (PCP) signaling system governs many aspects of polarized cell behavior. Here, we use an in vivo model of vertebrate mucociliary epithelial development to show that Dishevelled (Dvl) is essential for the apical positioning of basal bodies. We find that Dvl and Inturned mediate the activation of the Rho GTPase specifically at basal bodies, and that these three proteins together mediate the docking of basal bodies to the apical plasma membrane. Moreover, we find that the docking involves a Dvl-dependent association of basal bodies with membrane-bound vesicles and with the vesicle-trafficking protein, Sec8. Once docked, Dvl and Rho are once again required for the planar polarization of basal bodies that underlies directional beating of cilia. These results demonstrate novel functions for PCP signaling components and suggest that a common signaling appratus governs both apical docking and planar polarization of basal bodies.
doi:10.1038/ng.104
PMCID: PMC2771675  PMID: 18552847
24.  The Formin DAAM Functions as Molecular Effector of the Planar Cell Polarity Pathway during Axonal Development in Drosophila 
The Journal of Neuroscience  2015;35(28):10154-10167.
Recent studies established that the planar cell polarity (PCP) pathway is critical for various aspects of nervous system development and function, including axonal guidance. Although it seems clear that PCP signaling regulates actin dynamics, the mechanisms through which this occurs remain elusive. Here, we establish a functional link between the PCP system and one specific actin regulator, the formin DAAM, which has previously been shown to be required for embryonic axonal morphogenesis and filopodia formation in the growth cone. We show that dDAAM also plays a pivotal role during axonal growth and guidance in the adult Drosophila mushroom body, a brain center for learning and memory. By using a combination of genetic and biochemical assays, we demonstrate that Wnt5 and the PCP signaling proteins Frizzled, Strabismus, and Dishevelled act in concert with the small GTPase Rac1 to activate the actin assembly functions of dDAAM essential for correct targeting of mushroom body axons. Collectively, these data suggest that dDAAM is used as a major molecular effector of the PCP guidance pathway. By uncovering a signaling system from the Wnt5 guidance cue to an actin assembly factor, we propose that the Wnt5/PCP navigation system is linked by dDAAM to the regulation of the growth cone actin cytoskeleton, and thereby growth cone behavior, in a direct way.
doi:10.1523/JNEUROSCI.3708-14.2015
PMCID: PMC4502256  PMID: 26180192
axon growth; dDAAM; Drosophila; formin; mushroom body; PCP
25.  Observing planar cell polarity in multiciliated mouse airway epithelial cells 
Methods in cell biology  2015;127:37-54.
The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type.
doi:10.1016/bs.mcb.2015.01.016
PMCID: PMC4423753  PMID: 25837385

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