Primary cilia: cellular sensors for the skeleton.

PubMed

Anderson, Charles T; Castillo, Alesha B; Brugmann, Samantha A; Helms, Jill A; Jacobs, Christopher R; Stearns, Tim

2008-09-01

The primary cilium is a solitary, immotile cilium that is present in almost every mammalian cell type. Primary cilia are thought to function as chemosensors, mechanosensors, or both, depending on cell type, and have been linked to several developmental signaling pathways. Primary cilium malfunction has been implicated in several human diseases, the symptoms of which include vision and hearing loss, polydactyly, and polycystic kidneys. Recently, primary cilia have also been implicated in the development and homeostasis of the skeleton. In this review, we discuss the structure and formation of the primary cilium and some of the mechanical and chemical signals to which it could be sensitive, with a focus on skeletal biology. We also raise several unanswered questions regarding the role of primary cilia as mechanosensors and chemosensors and identify potential research avenues to address these questions.

Primary Cilia: Cellular Sensors for the Skeleton

PubMed Central

Anderson, Charles T.; Castillo, Alesha B.; Brugmann, Samantha A.; Helms, Jill A.; Jacobs, Christopher R.; Stearns, Tim

2010-01-01

The primary cilium is a solitary, immotile cilium that is present in almost every mammalian cell type. Primary cilia are thought to function as chemosensors, mechanosensors, or both, depending on cell type, and have been linked to several developmental signaling pathways. Primary cilium malfunction has been implicated in several human diseases, the symptoms of which include vision and hearing loss, polydactyly, and polycystic kidneys. Recently, primary cilia have also been implicated in the development and homeostasis of the skeleton. In this review, we discuss the structure and formation of the primary cilium and some of the mechanical and chemical signals to which it could be sensitive, with a focus on skeletal biology. We also raise several unanswered questions regarding the role of primary cilia as mechanosensors and chemosensors and identify potential research avenues to address these questions. PMID:18727074

Comparative study of the primary cilia in thyrocytes of adult mammals

PubMed Central

Utrilla, J C; Gordillo-Martínez, F; Gómez-Pascual, A; Fernández-Santos, J M; Garnacho, C; Vázquez-Román, V; Morillo-Bernal, J; García-Marín, R; Jiménez-García, A; Martín-Lacave, I

2015-01-01

Since their discovery in different human tissues by Zimmermann in 1898, primary cilia have been found in the vast majority of cell types in vertebrates. Primary cilia are considered to be cellular antennae that occupy an ideal cellular location for the interpretation of information both from the environment and from other cells. To date, in mammalian thyroid gland, primary cilia have been found in the thyrocytes of humans and dogs (fetuses and adults) and in rat embryos. The present study investigated whether the existence of this organelle in follicular cells is a general event in the postnatal thyroid gland of different mammals, using both immunolabeling by immunofluorescence and electron microscopy. Furthermore, we aimed to analyse the presence of primary cilia in various thyroid cell lines. According to our results, primary cilia are present in the adult thyroid gland of most mammal species we studied (human, pig, guinea pig and rabbit), usually as a single copy per follicular cell. Strikingly, they were not found in rat or mouse thyroid tissues. Similarly, cilia were also observed in all human thyroid cell lines tested, both normal and neoplastic follicular cells, but not in cultured thyrocytes of rat origin. We hypothesize that primary cilia could be involved in the regulation of normal thyroid function through specific signaling pathways. Nevertheless, further studies are needed to shed light on the permanence of these organelles in the thyroid gland of most species during postnatal life. PMID:26228270

Cilia/Ift protein and motor -related bone diseases and mouse models.

PubMed

Yuan, Xue; Yang, Shuying

2015-01-01

Primary cilia are essential cellular organelles projecting from the cell surface to sense and transduce developmental signaling. They are tiny but have complicated structures containing microtubule (MT)-based internal structures (the axoneme) and mother centriole formed basal body. Intraflagellar transport (Ift) operated by Ift proteins and motors are indispensable for cilia formation and function. Mutations in Ift proteins or Ift motors cause various human diseases, some of which have severe bone defects. Over the last few decades, major advances have occurred in understanding the roles of these proteins and cilia in bone development and remodeling by examining cilia/Ift protein-related human diseases and establishing mouse transgenic models. In this review, we describe current advances in the understanding of the cilia/Ift structure and function. We further summarize cilia/Ift-related human diseases and current mouse models with an emphasis on bone-related phenotypes, cilia morphology, and signaling pathways.

Primary Cilia and Dendritic Spines: Different but Similar Signaling Compartments

PubMed Central

Nechipurenko, Inna V.; Doroquez, David B.; Sengupta, Piali

2013-01-01

Primary non-motile cilia and dendritic spines are cellular compartments that are specialized to sense and transduce environmental cues and presynaptic signals, respectively. Despite their unique cellular roles, both compartments exhibit remarkable parallels in the general principles, as well as molecular mechanisms, by which their protein composition, membrane domain architecture, cellular interactions, and structural and functional plasticity are regulated. We compare and contrast the pathways required for the generation and function of cilia and dendritic spines, and suggest that insights from the study of one may inform investigations into the other of these critically important signaling structures. PMID:24048681

The Zn finger protein Iguana impacts Hedgehog signaling by promoting ciliogenesis.

PubMed

Glazer, Andrew M; Wilkinson, Alex W; Backer, Chelsea B; Lapan, Sylvain W; Gutzman, Jennifer H; Cheeseman, Iain M; Reddien, Peter W

2010-01-01

Hedgehog signaling is critical for metazoan development and requires cilia for pathway activity. The gene iguana was discovered in zebrafish as required for Hedgehog signaling, and encodes a novel Zn finger protein. Planarians are flatworms with robust regenerative capacities and utilize epidermal cilia for locomotion. RNA interference of Smed-iguana in the planarian Schmidtea mediterranea caused cilia loss and failure to regenerate new cilia, but did not cause defects similar to those observed in hedgehog(RNAi) animals. Smed-iguana gene expression was also similar in pattern to the expression of multiple other ciliogenesis genes, but was not required for expression of these ciliogenesis genes. iguana-defective zebrafish had too few motile cilia in pronephric ducts and in Kupffer's vesicle. Kupffer's vesicle promotes left-right asymmetry and iguana mutant embryos had left-right asymmetry defects. Finally, human Iguana proteins (dZIP1 and dZIP1L) localize to the basal bodies of primary cilia and, together, are required for primary cilia formation. Our results indicate that a critical and broadly conserved function for Iguana is in ciliogenesis and that this function has come to be required for Hedgehog signaling in vertebrates.

Microgravity induces inhibition of osteoblastic differentiation and mineralization through abrogating primary cilia.

PubMed

Shi, Wengui; Xie, Yanfang; He, Jinpeng; Zhou, Jian; Gao, Yuhai; Wei, Wenjun; Ding, Nan; Ma, Huiping; Xian, Cory J; Chen, Keming; Wang, Jufang

2017-05-12

It is well documented that microgravity in space environment leads to bone loss in astronauts. These physiological changes have also been validated by human and animal studies and modeled in cell-based analogs. However, the underlying mechanisms are elusive. In the current study, we identified a novel phenomenon that primary cilia (key sensors and functioning organelles) of rat calvarial osteoblasts (ROBs) gradually shrank and disappeared almost completely after exposure to simulated microgravity generated by a random positioning machine (RPM). Along with the abrogation of primary cilia, the differentiation, maturation and mineralization of ROBs were inhibited. We also found that the disappearance of primary cilia was prevented by treating ROBs with cytochalasin D, but not with LiCl or dynein light chain Tctex-type 1 (Dynlt1) siRNA. The repression of the differentiation, maturation and mineralization of ROBs was effectively offset by cytochalasin D treatment in microgravity conditions. Blocking ciliogenesis using intraflagellar transport protein 88 (IFT88) siRNA knockdown inhibited the ability of cytochalasin D to counteract this reduction of osteogenesis. These results indicate that the abrogation of primary cilia may be responsible for the microgravity's inhibition on osteogenesis. Reconstruction of primary cilia may become a potential strategy against bone loss induced by microgravity.

Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease.

PubMed

Lewis, Wesley R; Malarkey, Erik B; Tritschler, Douglas; Bower, Raqual; Pasek, Raymond C; Porath, Jonathan D; Birket, Susan E; Saunier, Sophie; Antignac, Corinne; Knowles, Michael R; Leigh, Margaret W; Zariwala, Maimoona A; Challa, Anil K; Kesterson, Robert A; Rowe, Steven M; Drummond, Iain A; Parant, John M; Hildebrandt, Friedhelm; Porter, Mary E; Yoder, Bradley K; Berbari, Nicolas F

2016-07-01

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.

Type I collagen-induced YAP nuclear expression promotes primary cilia growth and contributes to cell migration in confluent mouse embryo fibroblast 3T3-L1 cells.

PubMed

Xu, Qian; Liu, Xiaoling; Liu, Weiwei; Hayashi, Toshihiko; Yamato, Masayuki; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2018-05-30

The extracellular matrix (ECM) is a major biomechanical environment for all cells in vivo, and tightly controls wound healing and cancer progression. Type I collagen (Col I) is the most abundant component in ECM and plays an essential role for cell motility control and migration beyond structural support. Our previous results showed that Col I increased the length of primary cilia and the expression of primary cilia-associated proteins in 3T3-L1 cells. The Hippo/YAP pathway serves as a major integrator of cell surface-mediated signals and regulates key processes for the development and maintenance of tissue functions. In this study, we investigated the role of Hippo/YAP signaling in primary cilia growth of cells cultured on Col I-coated plate, as well as the potential link between primary cilia and migration. At 2-day post-confluence, YAP localization in the nucleus was dramatically increased when the cells were cultured on Col I-coated plate, accompanied by cilia growth. YAP inhibitor verteporfin repressed the growth of primary cilia as well as the expressions of ciliogenesis-associated proteins in confluent 3T3-L1 cells cultured on Col I-coated plate. Moreover, knockdown of either YAP or IFT88, one of the ciliogenesis-associated proteins, reversed the migration of confluent 3T3-L1 cells promoted by Col I-coating. In conclusion, activation of YAP pathway by Col I-coating of culture plate for confluent 3T3-L1 cells is positively associated with the primary cilia growth, which eventually results in promoted migration.

Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyoshi, Ko, E-mail: miyoshi@cc.okayama-u.ac.jp; Kasahara, Kyosuke; Miyazaki, Ikuko

2009-10-30

The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed withmore » Li{sub 2}CO{sub 3} were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.« less

Ultrastructural characterization of primary cilia in pathologically characterized human glioblastoma multiforme (GBM) tumors.

PubMed

Moser, Joanna J; Fritzler, Marvin J; Rattner, Jerome B

2014-01-01

Primary cilia are non-motile sensory cytoplasmic organelles that are involved in cell cycle progression. Ultrastructurally, the primary cilium region is complex, with normal ciliogenesis progressing through five distinct morphological stages in human astrocytes. Defects in early stages of ciliogenesis are key features of astrocytoma/glioblastoma cell lines and provided the impetus for the current study which describes the morphology of primary cilia in molecularly characterized human glioblastoma multiforme (GBM) tumors. Seven surgically resected human GBM tissue samples were molecularly characterized according to IDH1/2 mutation status, EGFR amplification status and MGMT promoter methylation status and were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. We report for the first time that primary cilia are disrupted in the early stages of ciliogenesis in human GBM tumors. We confirm that immature primary cilia and basal bodies/centrioles have aberrant ciliogenesis characteristics including absent paired vesicles, misshaped/swollen vesicular hats, abnormal configuration of distal appendages, and discontinuity of centriole microtubular blades. Additionally, the transition zone plate is able to form in the absence of paired vesicles on the distal end of the basal body and when a cilium progresses beyond the early stages of ciliogenesis, it has electron dense material clumped along the transition zone and a darkening of the microtubules at the proximal end of the cilium. Primary cilia play a role in a variety of human cancers. Previously primary cilia structure was perturbed in cultured cell lines derived from astrocytomas/glioblastomas; however there was always some question as to whether these findings were a cell culture phenomena. In this study we confirm that disruptions in ciliogenesis at early stages do occur in GBM tumors and that these ultrastructural findings bear resemblance to those previously observed in cell cultures. This is the first study to demonstrate that defects in cilia expression and function are a true hallmark of GBM tumors and correlate with their unrestrained growth. A review of the current ultrastructural profiles in the literature provides suggestions as to the best possible candidate protein that underlies defects in the early stages of ciliogenesis within GBM tumors.

CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms

PubMed Central

Panizzi, Jennifer R.; Becker-Heck, Anita; Castleman, Victoria H.; Al-Mutairi, Dalal; Liu, Yan; Loges, Niki T.; Pathak, Narendra; Austin-Tse, Christina; Sheridan, Eamonn; Schmidts, Miriam; Olbrich, Heike; Werner, Claudius; Häffner, Karsten; Hellman, Nathan; Chodhari, Rahul; Gupta, Amar; Kramer-Zucker, Albrecht; Olale, Felix; Burdine, Rebecca D.; Schier, Alexander F.; O’Callaghan, Christopher; Chung, Eddie MK; Reinhardt, Richard; Mitchison, Hannah M.; King, Stephen M.; Omran, Heymut; Drummond, Iain A.

2012-01-01

Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation, and to establish laterality1. Cilia motility defects cause Primary Ciliary Dyskinesia (PCD, MIM 242650), a disorder affecting 1:15-30,000 births. Cilia motility requires the assembly of multisubunit dynein arms that drive cilia bending2. Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD linked loci3. Here we show that the zebrafish cilia paralysis mutant schmalhanstn222 (smh) mutant encodes the coiled-coil domain containing 103 protein (Ccdc103), a foxj1a regulated gene. Screening 146 unrelated PCD families identified patients in six families with reduced outer dynein arms, carrying mutations in CCDC103. Dynein arm assembly in smh mutant zebrafish was rescued by wild-type but not mutant human CCDC103. Chlamydomonas Ccdc103 functions as a tightly bound, axoneme-associated protein. The results identify Ccdc103 as a novel dynein arm attachment factor that when mutated causes Primary Ciliary Dyskinesia. PMID:22581229

Primary Cilia as a Possible Link between Left-Right Asymmetry and Neurodevelopmental Diseases.

PubMed

Trulioff, Andrey; Ermakov, Alexander; Malashichev, Yegor

2017-01-25

Cilia have multiple functions in the development of the entire organism, and participate in the development and functioning of the central nervous system. In the last decade, studies have shown that they are implicated in the development of the visceral left-right asymmetry in different vertebrates. At the same time, some neuropsychiatric disorders, such as schizophrenia, autism, bipolar disorder, and dyslexia, are known to be associated with lateralization failure. In this review, we consider possible links in the mechanisms of determination of visceral asymmetry and brain lateralization, through cilia. We review the functions of seven genes associated with both cilia, and with neurodevelopmental diseases, keeping in mind their possible role in the establishment of the left-right brain asymmetry.

Primary Cilia as a Possible Link between Left-Right Asymmetry and Neurodevelopmental Diseases

PubMed Central

Trulioff, Andrey; Ermakov, Alexander; Malashichev, Yegor

2017-01-01

Cilia have multiple functions in the development of the entire organism, and participate in the development and functioning of the central nervous system. In the last decade, studies have shown that they are implicated in the development of the visceral left-right asymmetry in different vertebrates. At the same time, some neuropsychiatric disorders, such as schizophrenia, autism, bipolar disorder, and dyslexia, are known to be associated with lateralization failure. In this review, we consider possible links in the mechanisms of determination of visceral asymmetry and brain lateralization, through cilia. We review the functions of seven genes associated with both cilia, and with neurodevelopmental diseases, keeping in mind their possible role in the establishment of the left-right brain asymmetry. PMID:28125008

Diverse Roles of Axonemal Dyneins in Drosophila Auditory Neuron Function and Mechanical Amplification in Hearing.

PubMed

Karak, Somdatta; Jacobs, Julie S; Kittelmann, Maike; Spalthoff, Christian; Katana, Radoslaw; Sivan-Loukianova, Elena; Schon, Michael A; Kernan, Maurice J; Eberl, Daniel F; Göpfert, Martin C

2015-11-26

Much like vertebrate hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila are motile and amplify the mechanical input of the ear. Because the neurons bear mechanosensory primary cilia whose microtubule axonemes display dynein arms, we hypothesized that their motility is powered by dyneins. Here, we describe two axonemal dynein proteins that are required for Drosophila auditory neuron function, localize to their primary cilia, and differently contribute to mechanical amplification in hearing. Promoter fusions revealed that the two axonemal dynein genes Dmdnah3 (=CG17150) and Dmdnai2 (=CG6053) are expressed in chordotonal neurons, including the auditory ones in the fly's ear. Null alleles of both dyneins equally abolished electrical auditory neuron responses, yet whereas mutations in Dmdnah3 facilitated mechanical amplification, amplification was abolished by mutations in Dmdnai2. Epistasis analysis revealed that Dmdnah3 acts downstream of Nan-Iav channels in controlling the amplificatory gain. Dmdnai2, in addition to being required for amplification, was essential for outer dynein arms in auditory neuron cilia. This establishes diverse roles of axonemal dyneins in Drosophila auditory neuron function and links auditory neuron motility to primary cilia and axonemal dyneins. Mutant defects in sperm competition suggest that both dyneins also function in sperm motility.

Distinct patterns of primary and motile cilia in Rathke's cleft cysts and craniopharyngioma subtypes.

PubMed

Coy, Shannon; Du, Ziming; Sheu, Shu-Hsien; Woo, Terri; Rodriguez, Fausto J; Kieran, Mark W; Santagata, Sandro

2016-12-01

Cilia are highly conserved organelles, which serve critical roles in development and physiology. Motile cilia are expressed in a limited range of tissues, where they principally regulate local extracellular fluid dynamics. In contrast, primary cilia are expressed by many vertebrate cell types during interphase, and are intimately involved in the cell cycle and signal transduction. Notably, primary cilia are essential for vertebrate hedgehog pathway activity. Improved detection of motile cilia may assist in the diagnosis of some pathologic entities such as Rathke's cleft cysts, whereas characterizing primary cilia in neoplastic tissues may implicate cilia-dependent signaling pathways as critical for tumorigenesis. We show that immunohistochemistry for the nuclear transcription factor FOXJ1, a master regulator of motile ciliogenesis, robustly labels the motile ciliated epithelium of Rathke's cleft cysts. FOXJ1 expression discriminates Rathke's cleft cysts from entities in the sellar/suprasellar region with overlapping histologic features such as craniopharyngiomas. Co-immunohistochemistry for FOXJ1 and markers that highlight motile cilia such as acetylated tubulin (TUBA4A) and the small GTPase ARL13B further enhance the ability to identify diagnostic epithelial cells. In addition to highlighting motile cilia, ARL13B immunohistochemistry also robustly highlights primary cilia in formalin-fixed paraffin-embedded sections. Primary cilia are present throughout the neoplastic epithelium of adamantinomatous craniopharyngioma, but are limited to basally oriented cells near the fibrovascular stroma in papillary craniopharyngioma. Consistent with this differing pattern of primary ciliation, adamantinomatous craniopharyngiomas express significantly higher levels of SHH, and downstream targets such as PTCH1 and GLI2, compared with papillary craniopharyngiomas. In conclusion, motile ciliated epithelium can be readily identified using immunohistochemistry for FOXJ1, TUBA4A, and ARL13B, facilitating the diagnosis of Rathke's cleft cysts. Primary cilia can be identified by ARL13B immunohistochemistry in routine pathology specimens. The widespread presence of primary cilia in adamantinomatous craniopharyngioma implicates cilia-dependent hedgehog signaling in the pathogenesis of adamantinomatous craniopharyngioma.

Distinct Patterns of Primary and Motile Cilia in Rathke’s Cleft Cysts and Craniopharyngioma Subtypes

PubMed Central

Coy, Shannon; Du, Ziming; Sheu, Shu-Hsien; Woo, Terri; Rodriguez, Fausto J.; Kieran, Mark W.; Santagata, Sandro

2017-01-01

Cilia are highly conserved organelles which serve critical roles in development and physiology. Motile cilia are expressed in a limited range of tissues, where they principally regulate local extracellular fluid dynamics. In contrast, primary cilia are expressed by many vertebrate cell types during interphase, and are intimately involved in the cell cycle and signal transduction. Notably, primary cilia are essential for vertebrate hedgehog pathway activity. Improved detection of motile cilia may assist in the diagnosis of some pathologic entities such as Rathke’s cleft cysts while characterizing primary cilia in neoplastic tissues may implicate cilia-dependent signaling pathways as critical for tumorigenesis. We show that immunohistochemistry for the nuclear transcription factor FOXJ1, a master regulator of motile ciliogenesis, robustly labels the motile ciliated epithelium of Rathke’s cleft cysts. FOXJ1 expression discriminates Rathke’s cleft cysts from entities in the sellar/suprasellar region with overlapping histologic features such as craniopharyngiomas. Co-immunohistochemistry for FOXJ1 and markers that highlight motile cilia such as acetylated tubulin (TUBA4A) and the small GTPase ARL13B further enhance the ability to identify diagnostic epithelial cells. In addition to highlighting motile cilia, ARL13B immunohistochemistry also robustly highlights primary cilia in formalin-fixed paraffin-embedded sections. Primary cilia are present throughout the neoplastic epithelium of adamantinomatous craniopharyngioma, but are limited to basally oriented cells near the fibrovascular stroma in papillary craniopharyngioma. Consistent with this differing pattern of primary ciliation, adamantinomatous craniopharyngiomas express significantly higher levels of SHH, and downstream targets such as PTCH1 and GLI2, compared to papillary craniopharyngiomas. In conclusion, motile ciliated epithelium can be readily identified using immunohistochemistry for FOXJ1, TUBA4A and ARL13B, facilitating the diagnosis of Rathke’s cleft cyst. Primary cilia can be identified by ARL13B immunohistochemistry in routine pathology specimens. The widespread presence of primary cilia in adamantinomatous craniopharyngioma implicates cilia-dependent hedgehog signaling in the pathogenesis of adamantinomatous craniopharyngioma. PMID:27562488

Silibinin negatively contributes to primary cilia length via autophagy regulated by histone deacetylase 6 in confluent mouse embryo fibroblast 3T3-L1 cells.

PubMed

Xu, Qian; Liu, Wei; Liu, Xiaoling; Liu, Weiwei; Wang, Hongju; Yao, Guodong; Zang, Linghe; Hayashi, Toshihiko; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2016-09-01

Primary cilium is a cellular antenna, signalling as a sensory organelle. Numerous pathological manifestation is associated with change of its length. Although the interaction between autophagy and primary cilia has been suggested, the role of autophagy in primary cilia length is largely unknown. In this study the primary cilia were immunostained and observed by using confocal fluorescence microscopy, and we found that silibinin, a natural flavonoid, shortened the length of primary cilia, meanwhile it also induced autophagy in 3T3-L1 cells. This study was designed to investigate the significance of silibinin-induced autophagy in primary ciliary structure in confluent mouse embryo fibroblast 3T3-L1 cells. Either blocking the autophagic flux with pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), or transfection of siRNA targeting LC3 inhibited the reduction of cilia length caused by silibinin exposure. Autophagy induced by silibinin decreased expressions of the cilia-associated proteins, such as IFT88, KIF3a and Ac-tubulin, while 3-MA restored it, indicating that autophagy induced by silibinin led to a reduction of primary cilia length. Histone deacetylase 6 (HDAC6), which was suggested as a mediator of autophagy, was up-regulated by silibinin in a time-dependent manner. In addition, 3T3-L1 cells treated with siRNA against HDAC6 had a reduced autophagic level and were protected from silibinin-induced cilia shortening. Taken together, we conclude that the HDAC6-mediated autophagy negatively regulates primary cilia length during silibinin treatment and has the potential to serve as a therapeutic target for primary cilia-associated ciliopathies. These findings thus provide new information about the potential link between autophagy and primary cilia.

Cystogenesis and elongated primary cilia in Tsc1-deficient distal convoluted tubules

PubMed Central

Armour, Eric A.; Carson, Robert P.

2012-01-01

Tuberous sclerosis complex (TSC) is a multiorgan hamartomatous disease caused by loss of function mutations of either the TSC1 or TSC2 genes. Neurological symptoms of TSC predominate in younger patients, but renal pathologies are a serious aspect of the disease in older children and adults. To study TSC pathogenesis in the kidney, we inactivated the mouse Tsc1 gene in the distal convoluted tubules (DCT). At young ages, Tsc1 conditional knockout (CKO) mice have enlarged kidneys and mild cystogenesis with increased mammalian target of rapamycin complex (mTORC)1 but decreased mTORC2 signaling. Treatment with the mTORC1 inhibitor rapamycin reduces kidney size and cystogenesis. Rapamycin withdrawal led to massive cystogenesis involving both distal as well as proximal tubules. To assess the contribution of decreased mTORC2 signaling in kidney pathogenesis, we also generated Rictor CKO mice. These animals did not have any detectable kidney pathology. Finally, we examined primary cilia in the DCT. Cilia were longer in Tsc1 CKO mice, and rapamycin treatment returned cilia length to normal. Rictor CKO mice had normal cilia in the DCT. Overall, our findings suggest that loss of the Tsc1 gene in the DCT is sufficient for renal cystogenesis. This cytogenesis appears to be mTORC1 but not mTORC2 dependent. Intriguingly, the mechanism may be cell autonomous as well as non-cell autonomous and possibly involves the length and function of primary cilia. PMID:22674026

Cystogenesis and elongated primary cilia in Tsc1-deficient distal convoluted tubules.

PubMed

Armour, Eric A; Carson, Robert P; Ess, Kevin C

2012-08-15

Tuberous sclerosis complex (TSC) is a multiorgan hamartomatous disease caused by loss of function mutations of either the TSC1 or TSC2 genes. Neurological symptoms of TSC predominate in younger patients, but renal pathologies are a serious aspect of the disease in older children and adults. To study TSC pathogenesis in the kidney, we inactivated the mouse Tsc1 gene in the distal convoluted tubules (DCT). At young ages, Tsc1 conditional knockout (CKO) mice have enlarged kidneys and mild cystogenesis with increased mammalian target of rapamycin complex (mTORC)1 but decreased mTORC2 signaling. Treatment with the mTORC1 inhibitor rapamycin reduces kidney size and cystogenesis. Rapamycin withdrawal led to massive cystogenesis involving both distal as well as proximal tubules. To assess the contribution of decreased mTORC2 signaling in kidney pathogenesis, we also generated Rictor CKO mice. These animals did not have any detectable kidney pathology. Finally, we examined primary cilia in the DCT. Cilia were longer in Tsc1 CKO mice, and rapamycin treatment returned cilia length to normal. Rictor CKO mice had normal cilia in the DCT. Overall, our findings suggest that loss of the Tsc1 gene in the DCT is sufficient for renal cystogenesis. This cytogenesis appears to be mTORC1 but not mTORC2 dependent. Intriguingly, the mechanism may be cell autonomous as well as non-cell autonomous and possibly involves the length and function of primary cilia.

Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease

PubMed Central

Lewis, Wesley R.; Malarkey, Erik B.; Tritschler, Douglas; Bower, Raqual; Pasek, Raymond C.; Porath, Jonathan D.; Birket, Susan E.; Saunier, Sophie; Antignac, Corinne; Leigh, Margaret W.; Zariwala, Maimoona A.; Drummond, Iain A.; Parant, John M.; Hildebrandt, Friedhelm; Yoder, Bradley K.

2016-01-01

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or ‘primary’ cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic. PMID:27472056

The role of the cilium in hereditary tumor predisposition syndromes

PubMed Central

Klasson, Timothy D.; Giles, Rachel H.

2014-01-01

The primary cilium is a highly conserved cell organelle that is closely connected to processes involved in cell patterning and replication. Amongst their many functions, cilia act as “signal towers” through which cell-cell signaling cascades pass. Dysfunction of cilia or the myriad processes that are connected with cilium function can lead to disease. Due to the sheer number of cellular processes that at some point involve the primary cilium, the effects of misregulation are highly heterogeneous between different cell populations. However, because of the importance of primary cilia in the development, growth, patterning and orientation of cells and tissues, a common thread has emerged in which defective cilia can lead to disorganization, which can contribute to the growth of neoplasms, including cancer and pre-cancerous phenotypes. Because cilia are so vital for signaling during cell replication and the cell fate decisions that are important in childhood growth, symptoms often arise early in life. Here we review recent work connecting misregulation of the primary cilium with tumor formation in a variety of tissues in the developing body, with a particular focus on the syndromes in which classic tumor genes are mutated, including von Hippel-Lindau disease (OMIM 193300), adenomatous polyposis coli (OMIM 175100), tuberous sclerosis (OMIM 191100) and Birt-Hogg-Dubé syndrome (OMIM 135150). Timely diagnosis of these syndromes is essential for entry into appropriate screening protocols, which have been shown to effectively prolong life expectancy in these cohorts of patients. PMID:27625869

Can Tissue Cilia Lengths and Urine Cilia Proteins Be Markers of Kidney Diseases?

PubMed

Park, Kwon Moo

2018-05-01

The primary cilium is an organelle which consists of a microtubule in the core and a surrounding cilia membrane, and has long been recognized as a "vestigial organelle". However, new evidence demonstrates that the primary cilium has a notable effect on signal transduction in the cell and is associated with some genetic and non-genetic diseases. In the kidney, the primary cilium protrudes into the Bowman's space and the tubular lumen from the apical side of epithelial cells. The length of primary cilia is dynamically altered during the normal cell cycle, being shortened by retraction into the cell body at the entry of cell division and elongated at differentiation. Furthermore, the length of primary cilia is also dynamically changed in the cells, as a result and/or cause, during the progression of various kidney diseases including acute kidney injury and chronic kidney disease. Notably, recent data has demonstrated that the shortening of the primary cilium in the cell is associated with fragmentation, apart from retraction into the cell body, in the progression of diseases and that the fragmented primary cilia are released into the urine. This data reveals that the alteration of primary cilia length could be related to the progression of diseases. This review will consider if primary cilia length alteration is associated with the progression of kidney diseases and if the length of tissue primary cilia and the presence or increase of cilia proteins in the urine is indicative of kidney diseases.

HDAC6 inhibition suppresses chondrosarcoma by restoring the expression of primary cilia.

PubMed

Xiang, Wei; Guo, Fengjing; Cheng, Weiting; Zhang, Jiaming; Huang, Junming; Wang, Rui; Ma, Zhongxi; Xu, Kai

2017-07-01

Chondrosarcoma is a bone tumor characterized by the secretion of a cartilage-like extracellular matrix. It has been proved to lack extracellular sensor primary cilia. This study aimed to illustrate a feasible therapeutic method for chondrosarcoma by regulating primary cilia assembly through inhibiting histone deacetylases 6 (HDAC6) activation. In order to detect the interaction between primary cilia and HDAC6 in human chondrosarcoma, Tubastatin A and small interfering RNA (siRNA) were used to inhibit the endogenous expression of HDAC6. Cell viability test and Transwell assay were applied to evaluate the effects of malignant biological properties. Primary cilia staining and related proteins were detected. The abnormal expression of HDAC6 and cilia intraflagellar transport protein 88 (IFT88) was found in chondrosarcoma tissues. The inhibition of HDAC6 could downregulate the proliferation of chondrosarcoma cells in a concentration- and time-dependent manner and suppress the invasion capacity of tumor cells. Besides, the downregulation of HDAC6 exhibited a negative effect on the proliferation of relevant proteins but a positive effect on the primary cilia-related expression of IFT88 and acetylated α-tubulin. Primary cilia restoration could be observed after HDAC6 siRNA transfection. The Aurora A-HDAC6 cascade was involved in regulating primary cilia resorption by affecting α-tubulin deacetylation and Tubastatin A could inhibit chondrosarcoma cell growth in vivo. These results indicate that restricting HDAC6 can restore primary cilia assembly accompanied with suppressed chondrosarcoma cell proliferation and invasion capacities. Thus, promoting primary cilia restoration by targeting HDAC6 may be a feasible potential therapeutic method for chondro-sarcoma treatment.

Reduced primary cilia length and altered Arl13b expression are associated with deregulated chondrocyte Hedgehog signaling in alkaptonuria

PubMed Central

Thorpe, Stephen D.; Gambassi, Silvia; Thompson, Clare L.; Chandrakumar, Charmilie

2017-01-01

Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2‐dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog‐related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU. PMID:28158906

Systematic discovery of novel ciliary genes through functional genomics in the zebrafish

PubMed Central

Choksi, Semil P.; Babu, Deepak; Lau, Doreen; Yu, Xianwen; Roy, Sudipto

2014-01-01

Cilia are microtubule-based hair-like organelles that play many important roles in development and physiology, and are implicated in a rapidly expanding spectrum of human diseases, collectively termed ciliopathies. Primary ciliary dyskinesia (PCD), one of the most prevalent of ciliopathies, arises from abnormalities in the differentiation or motility of the motile cilia. Despite their biomedical importance, a methodical functional screen for ciliary genes has not been carried out in any vertebrate at the organismal level. We sought to systematically discover novel motile cilia genes by identifying the genes induced by Foxj1, a winged-helix transcription factor that has an evolutionarily conserved role as the master regulator of motile cilia biogenesis. Unexpectedly, we find that the majority of the Foxj1-induced genes have not been associated with cilia before. To characterize these novel putative ciliary genes, we subjected 50 randomly selected candidates to a systematic functional phenotypic screen in zebrafish embryos. Remarkably, we find that over 60% are required for ciliary differentiation or function, whereas 30% of the proteins encoded by these genes localize to motile cilia. We also show that these genes regulate the proper differentiation and beating of motile cilia. This collection of Foxj1-induced genes will be invaluable for furthering our understanding of ciliary biology, and in the identification of new mutations underlying ciliary disorders in humans. PMID:25139857

Myosin VIIa as a common component of cilia and microvilli.

PubMed

Wolfrum, U; Liu, X; Schmitt, A; Udovichenko, I P; Williams, D S

1998-01-01

The distribution of myosin VIIa, which is defective or absent in Usher syndrome 1B, was studied in a variety of tissues by immunomicroscopy. The primary aim was to determine whether this putative actin-based mechanoenzyme is a common component of cilia. Previously, it has been proposed that defective ciliary function might be the basis of some forms of Usher syndrome. Myosin VIIa was detected in cilia from cochlear hair cells, olfactory neurons, kidney distal tubules, and lung bronchi. It was also found to cofractionate with the axonemal fraction of retinal photoreceptor cells. Immunolabeling appeared most concentrated in the periphery of the transition zone of the cilia. This general presence of a myosin in cilia is surprising, given that cilia are dominated by microtubules, and not actin filaments. In addition to cilia, myosin VIIa was also found in actin-rich microvilli of different types of cell. We conclude that myosin VIIa is a common component of cilia and microvilli.

Orpk mouse model of polycystic kidney disease reveals essential role of primary cilia in pancreatic tissue organization.

PubMed

Cano, David A; Murcia, Noel S; Pazour, Gregory J; Hebrok, Matthias

2004-07-01

Polycystic kidney disease (PKD) includes a group of disorders that are characterized by the presence of cysts in the kidney and other organs, including the pancreas. Here we show that in orpk mice, a model system for PKD that harbors a mutation in the gene that encodes the polaris protein, pancreatic defects start to occur at the end of gestation, with an initial expansion of the developing pancreatic ducts. Ductal dilation continues rapidly after birth and results in the formation of large, interconnected cysts. Expansion of pancreatic ducts is accompanied by apoptosis of neighboring acinar cells, whereas endocrine cell differentiation and islet formation appears to be unaffected. Polaris has been shown to co-localize with primary cilia, and these structures have been implicated in the formation of renal cysts. In the orpk pancreas, cilia numbers are reduced and cilia length is decreased. Expression of polycystin-2, a protein involved in PKD, is mislocalized in orpk mice. Furthermore, the cellular localization of beta-catenin, a protein involved in cell adhesion and Wnt signaling, is altered. Thus, polaris and primary cilia function are required for the maturation and maintenance of proper tissue organization in the pancreas.

A compartmentalized phosphoinositide signaling axis at cilia is regulated by INPP5E to maintain cilia and promote Sonic Hedgehog medulloblastoma.

PubMed

Conduit, S E; Ramaswamy, V; Remke, M; Watkins, D N; Wainwright, B J; Taylor, M D; Mitchell, C A; Dyson, J M

2017-10-26

Sonic Hedgehog (SHH) signaling at primary cilia drives the proliferation and progression of a subset of medulloblastomas, the most common malignant paediatric brain tumor. Severe side effects associated with conventional treatments and resistance to targeted therapies has led to the need for new strategies. SHH signaling is dependent on primary cilia for signal transduction suggesting the potential for cilia destabilizing mechanisms as a therapeutic target. INPP5E is an inositol polyphosphate 5-phosphatase that hydrolyses PtdIns(4,5)P 2 and more potently, the phosphoinositide (PI) 3-kinase product PtdIns(3,4,5)P 3 . INPP5E promotes SHH signaling during embryonic development via PtdIns(4,5)P 2 hydrolysis at cilia, that in turn regulates the cilia recruitment of the SHH suppressor GPR161. However, the role INPP5E plays in cancer is unknown and the contribution of PI3-kinase signaling to cilia function is little characterized. Here, we reveal INPP5E promotes SHH signaling in SHH medulloblastoma by negatively regulating a cilia-compartmentalized PI3-kinase signaling axis that maintains primary cilia on tumor cells. Conditional deletion of Inpp5e in a murine model of constitutively active Smoothened-driven medulloblastoma slowed tumor progression, suppressed cell proliferation, reduced SHH signaling and promoted tumor cell cilia loss. PtdIns(3,4,5)P 3 , its effector pAKT and the target pGSK3β, which when non-phosphorylated promotes cilia assembly/stability, localized to tumor cell cilia. The number of PtdIns(3,4,5)P 3 /pAKT/pGSK3β-positive cilia was increased in cultured Inpp5e-null tumor cells relative to controls. PI3-kinase inhibition or expression of wild-type, but not catalytically inactive HA-INPP5E partially rescued cilia loss in Inpp5e-null tumor cells in vitro. INPP5E mRNA and copy number were reduced in human SHH medulloblastoma compared to other molecular subtypes and consistent with the murine model, reduced INPP5E was associated with improved overall survival. Therefore our study identifies a compartmentalized PtdIns(3,4,5)P 3 /AKT/GSK3β signaling axis at cilia in SHH-dependent medulloblastoma that is regulated by INPP5E to maintain tumor cell cilia, promote SHH signaling and thereby medulloblastoma progression.

Primary Cilia: Highly Sophisticated Biological Sensors

PubMed Central

Abou Alaiwi, Wissam A.; Lo, Shao T.; Nauli, Surya M.

2009-01-01

Primary cilia, thin hair-like structures protruding from the apical surface of most mammalian cells, have gained the attention of many researchers over the past decade. Primary cilia are microtubule-filled sensory organelles that are enclosed within the ciliary membrane. They originate at the cell surface from the mother centriole that becomes the mature basal body. In this review, we will discuss recent literatures on the roles of cilia as sophisticated sensory organelles. With particular emphasis on vascular endothelia and renal epithelia, the mechanosensory role of cilia in sensing fluid shear stress will be discussed. Also highlighted is the ciliary involvement in cell cycle regulation, development, cell signaling and cancer. Finally, primary cilia-related disorders will be briefly described. PMID:22423203

Reduced primary cilia length and altered Arl13b expression are associated with deregulated chondrocyte Hedgehog signaling in alkaptonuria.

PubMed

Thorpe, Stephen D; Gambassi, Silvia; Thompson, Clare L; Chandrakumar, Charmilie; Santucci, Annalisa; Knight, Martin M

2017-09-01

Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2-dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog-related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU. © 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.

Primary Cilia Are Not Calcium-Responsive Mechanosensors

PubMed Central

Delling, M.; Indzhykulian, A. A.; Liu, X.; Liu, Y.; Xie, T.; Corey, D. P.; Clapham, D. E.

2016-01-01

Primary cilia are solitary, generally non-motile, hair-like protrusions that extend from the surface of cells between cell divisions. Their antenna-like structure leads naturally to the assumption that they sense the surrounding environment, the most common hypothesis being sensation of mechanical force through calcium-permeable ion channels within the cilium1. This Ca2+- Responsive MechanoSensor (CaRMS) hypothesis for primary cilia has been invoked to explain a large range of biological responses, from control of left-right axis determination in embryonic development to adult progression of polycystic kidney disease and some cancers2,3. Here, we report the complete lack of mechanically induced calcium increases in primary cilia, in tissues upon which this hypothesis has been based. First, we developed a transgenic mouse, Arl13b-mCherry-GECO1.2, expressing a ratiometric genetically encoded calcium indicator (GECI) in all primary cilia. We then measured responses to flow in primary cilia of cultured kidney epithelial cells, kidney thick ascending tubules, crown cells of the embryonic node, kinocilia of inner ear hair cells, and several cell lines. Cilia-specific Ca2+ influxes were not observed in physiological or even highly supraphysiological levels of fluid flow. We conclude that mechanosensation, if it originates in primary cilia, is not via calcium signaling. PMID:27007841

Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells

NASA Technical Reports Server (NTRS)

Nauli, Surya M.; Alenghat, Francis J.; Luo, Ying; Williams, Eric; Vassilev, Peter; Li, Xiaogang; Elia, Andrew E H.; Lu, Weining; Brown, Edward M.; Quinn, Stephen J.;

Primary Cilium-Regulated EG-VEGF Signaling Facilitates Trophoblast Invasion.

PubMed

Wang, Chia-Yih; Tsai, Hui-Ling; Syu, Jhih-Siang; Chen, Ting-Yu; Su, Mei-Tsz

2017-06-01

Trophoblast invasion is an important event in embryo implantation and placental development. During these processes, endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is the key regulator mediating the crosstalk at the feto-maternal interface. The primary cilium is a cellular antenna receiving environmental signals and is crucial for proper development. However, little is known regarding the role of the primary cilium in early human pregnancy. Here, we demonstrate that EG-VEGF regulates trophoblast cell invasion via primary cilia. We found that EG-VEGF activated ERK1/2 signaling and subsequent upregulation of MMP2 and MMP9, thereby facilitating cell invasion in human trophoblast HTR-8/SVneo cells. Inhibition of ERK1/2 alleviated the expression of MMPs and trophoblast cell invasion after EG-VEGF treatment. In addition, primary cilia were observed in all the trophoblast cell lines tested and, more importantly, in human first-trimester placental tissue. The receptor of EG-VEGF, PROKR1, was detected in primary cilia. Depletion of IFT88, the intraflagellar transporter required for ciliogenesis, inhibited primary cilium growth, thereby ameliorating ERK1/2 activation, MMP upregulation, and trophoblast cell invasion promoted by EG-VEGF. These findings demonstrate a novel function of primary cilia in controlling EG-VEGF-regulated trophoblast invasion and reveal the underlying molecular mechanism. J. Cell. Physiol. 232: 1467-1477, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Odf2-deficient mother centrioles lack distal/subdistal appendages and the ability to generate primary cilia.

PubMed

Ishikawa, Hiroaki; Kubo, Akiharu; Tsukita, Shoichiro; Tsukita, Sachiko

2005-05-01

Outer dense fibre 2 (Odf2; also known as cenexin) was initially identified as a main component of the sperm tail cytoskeleton, but was later shown to be a general scaffold protein that is specifically localized at the distal/subdistal appendages of mother centrioles. Here we show that Odf2 expression is suppressed in mouse F9 cells when both alleles of Odf2 genes are deleted. Unexpectedly, the cell cycle of Odf2(-/-) cells does not seem to be affected. Immunofluorescence and ultrathin-section electron microscopy reveals that in Odf2(-/-) cells, distal/subdistal appendages disappear from mother centrioles, making it difficult to distinguish mother from daughter centrioles. In Odf2(-/-) cells, however, the formation of primary cilia is completely suppressed, although approximately 25% of wild-type F9 cells are ciliated under the steady-state cell cycle. The loss of primary cilia in Odf2(-/-) F9 cells can be rescued by exogenous Odf2 expression. These findings indicate that Odf2 is indispensable for the formation of distal/subdistal appendages and the generation of primary cilia, but not for other cell-cycle-related centriolar functions.

Identification of BCAP, a new protein associated with basal bodies and centrioles.

PubMed

Ponsard, Cecile; Seltzer, Virginie; Perret, Eric; Tournier, Frederic; Middendorp, Sandrine

2007-05-01

Cilia exert critical functions in numerous organisms, including that of cell motility, fluid transport and protozoan locomotion. Defects in this organelle can lead to lethal pathologies in humans, including primary ciliary dyskinesia. An understanding of the cilia formation process would lead to better characterization of defects involved in such pathologies. In the present study, we identified a gene encoding a novel human protein, BCAP for Basal body Centriole-Associated Protein, which shares homologies with a previously described protein, Outer Dense Fiber 2 (ODF2). ODF2, a major component of the sperm tail cytoskeleton, is required for the formation of mother centriole distal/subdistal appendages and the generation of primary cilia. Here, we show that the bcap gene contains 18 alternatively spliced exons and encodes five different isoforms, three long and two short ones. BCAP is preferentially expressed in cilia/flagella containing tissues. Moreover, its expression is correlated with cilia formation during mucociliary differentiation of human nasal epithelial cells. Using immunofluorescence analyses, BCAP was localized within basal bodies of ciliated cells and within centrioles of proliferating cells. In light of the several spliced isoforms of BCAP and the particular localization of the protein, BCAP isoforms could play distinct roles in cilia and in centrosomes.

Culture of Primary Ciliary Dyskinesia Epithelial Cells at Air-Liquid Interface Can Alter Ciliary Phenotype but Remains a Robust and Informative Diagnostic Aid

PubMed Central

Coles, Janice L.; Williams, Gwyneth; Rutman, Andrew; Goggin, Patricia M.; Adam, Elizabeth C.; Page, Anthony; Evans, Hazel J.; Lackie, Peter M.; O’Callaghan, Christopher; Lucas, Jane S.

2014-01-01

Background The diagnosis of primary ciliary dyskinesia (PCD) requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI) aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns. Methods We retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD n = 111) was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture. Results Ciliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced. Conclusions The change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia. PMID:24586956

Reptin/Ruvbl2 is a Lrrc6/Seahorse interactor essential for cilia motility

PubMed Central

Zhao, Lu; Yuan, Shiaulou; Cao, Ying; Kallakuri, Sowjanya; Li, Yuanyuan; Kishimoto, Norihito; DiBella, Linda; Sun, Zhaoxia

2013-01-01

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease caused by defective cilia motility. The identified PCD genes account for about half of PCD incidences and the underlying mechanisms remain poorly understood. We demonstrate that Reptin/Ruvbl2, a protein known to be involved in epigenetic and transcriptional regulation, is essential for cilia motility in zebrafish. We further show that Reptin directly interacts with the PCD protein Lrrc6/Seahorse and this interaction is critical for the in vivo function of Lrrc6/Seahorse in zebrafish. Moreover, whereas the expression levels of multiple dynein arm components remain unchanged or become elevated, the density of axonemal dynein arms is reduced in reptinhi2394 mutants. Furthermore, Reptin is highly enriched in the cytosol and colocalizes with Lrrc6/Seahorse. Combined, these results suggest that the Reptin-Lrrc6/Seahorse complex is involved in dynein arm formation. We also show that although the DNA damage response is induced in reptinhi2394 mutants, it remains unchanged in cilia biogenesis mutants and lrrc6/seahorse mutants, suggesting that increased DNA damage response is not intrinsic to ciliary defects and that in vertebrate development, Reptin functions in multiple processes, both cilia specific and cilia independent. PMID:23858445

Reptin/Ruvbl2 is a Lrrc6/Seahorse interactor essential for cilia motility.

PubMed

Zhao, Lu; Yuan, Shiaulou; Cao, Ying; Kallakuri, Sowjanya; Li, Yuanyuan; Kishimoto, Norihito; DiBella, Linda; Sun, Zhaoxia

2013-07-30

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease caused by defective cilia motility. The identified PCD genes account for about half of PCD incidences and the underlying mechanisms remain poorly understood. We demonstrate that Reptin/Ruvbl2, a protein known to be involved in epigenetic and transcriptional regulation, is essential for cilia motility in zebrafish. We further show that Reptin directly interacts with the PCD protein Lrrc6/Seahorse and this interaction is critical for the in vivo function of Lrrc6/Seahorse in zebrafish. Moreover, whereas the expression levels of multiple dynein arm components remain unchanged or become elevated, the density of axonemal dynein arms is reduced in reptin(hi2394) mutants. Furthermore, Reptin is highly enriched in the cytosol and colocalizes with Lrrc6/Seahorse. Combined, these results suggest that the Reptin-Lrrc6/Seahorse complex is involved in dynein arm formation. We also show that although the DNA damage response is induced in reptin(hi2394) mutants, it remains unchanged in cilia biogenesis mutants and lrrc6/seahorse mutants, suggesting that increased DNA damage response is not intrinsic to ciliary defects and that in vertebrate development, Reptin functions in multiple processes, both cilia specific and cilia independent.

hemingway is required for sperm flagella assembly and ciliary motility in Drosophila.

PubMed

Soulavie, Fabien; Piepenbrock, David; Thomas, Joëlle; Vieillard, Jennifer; Duteyrat, Jean-Luc; Cortier, Elisabeth; Laurençon, Anne; Göpfert, Martin C; Durand, Bénédicte

2014-04-01

Cilia play major functions in physiology and development, and ciliary dysfunctions are responsible for several diseases in humans called ciliopathies. Cilia motility is required for cell and fluid propulsion in organisms. In humans, cilia motility deficiencies lead to primary ciliary dyskinesia, with upper-airways recurrent infections, left-right asymmetry perturbations, and fertility defects. In Drosophila, we identified hemingway (hmw) as a novel component required for motile cilia function. hmw encodes a 604-amino acid protein characterized by a highly conserved coiled-coil domain also found in the human orthologue, KIAA1430. We show that HMW is conserved in species with motile cilia and that, in Drosophila, hmw is expressed in ciliated sensory neurons and spermatozoa. We created hmw-knockout flies and found that they are hearing impaired and male sterile. hmw is implicated in the motility of ciliated auditory sensory neurons and, in the testis, is required for elongation and maintenance of sperm flagella. Because HMW is absent from mature flagella, we propose that HMW is not a structural component of the motile axoneme but is required for proper acquisition of motile properties. This identifies HMW as a novel, evolutionarily conserved component necessary for motile cilium function and flagella assembly.

The ciliopathy gene Rpgrip1l is essential for hair follicle development.

PubMed

Chen, Jiang; Laclef, Christine; Moncayo, Alejandra; Snedecor, Elizabeth R; Yang, Ning; Li, Li; Takemaru, Ken-Ichi; Paus, Ralf; Schneider-Maunoury, Sylvie; Clark, Richard A

2015-03-01

The primary cilium is essential for skin morphogenesis through regulating the Notch, Wnt, and hedgehog signaling pathways. Prior studies on the functions of primary cilia in the skin were based on the investigations of genes that are essential for cilium formation. However, none of these ciliogenic genes has been linked to ciliopathy, a group of disorders caused by abnormal formation or function of cilia. To determine whether there is a genetic and molecular link between ciliopathies and skin morphogenesis, we investigated the role of RPGRIP1L, a gene mutated in Joubert (JBTS) and Meckel (MKS) syndromes, two severe forms of ciliopathy, in the context of skin development. We found that RPGRIP1L is essential for hair follicle morphogenesis. Specifically, disrupting the Rpgrip1l gene in mice resulted in reduced proliferation and differentiation of follicular keratinocytes, leading to hair follicle developmental defects. These defects were associated with significantly decreased primary cilium formation and attenuated hedgehog signaling. In contrast, we found that hair follicle induction and polarization and the development of interfollicular epidermis were unaffected. This study indicates that RPGRIP1L, a ciliopathy gene, is essential for hair follicle morphogenesis likely through regulating primary cilia formation and the hedgehog signaling pathway.

Cytoskeleton-related regulation of primary cilia shortening mediated by melanin-concentrating hormone receptor 1.

PubMed

Tomoshige, Sakura; Kobayashi, Yuki; Hosoba, Kosuke; Hamamoto, Akie; Miyamoto, Tatsuo; Saito, Yumiko

2017-11-01

Primary cilia are specialized microtubule-based organelles. Their importance is highlighted by the gamut of ciliary diseases associated with various syndromes including diabetes and obesity. Primary cilia serve as signaling hubs through selective interactions with ion channels and conventional G-protein-coupled receptors (GPCRs). Melanin-concentrating hormone (MCH) receptor 1 (MCHR1), a key regulator of feeding, is selectively expressed in neuronal primary cilia in distinct regions of the mouse brain. We previously found that MCH acts on ciliary MCHR1 and induces cilia shortening through a Gi/o-dependent Akt pathway with no cell cycle progression. Many factors can participate in cilia length control. However, the mechanisms for how these molecules are relocated and coordinated to activate cilia shortening are poorly understood. In the present study, we investigated the role of cytoskeletal dynamics in regulating MCH-induced cilia shortening using clonal MCHR1-expressing hTERT-RPE1 cells. Pharmacological and biochemical approaches showed that cilia shortening mediated by MCH was associated with increased soluble cytosolic tubulin without changing the total tubulin amount. Enhanced F-actin fiber intensity was also observed in MCH-treated cells. The actions of various pharmacological agents revealed that coordinated actin machinery, especially actin polymerization, was required for MCHR1-mediated cilia shortening. A recent report indicated the existence of actin-regulated machinery for cilia shortening through GPCR agonist-dependent ectosome release. However, our live-cell imaging experiments showed that MCH progressively elicited cilia shortening without exclusion of fluorescence-positive material from the tip. Short cilia phenotypes have been associated with various metabolic disorders. Thus, the present findings may contribute toward better understanding of how the cytoskeleton is involved in the GPCR ligand-triggered cilia shortening with cell mechanical properties that underlies clinical manifestations such as obesity. Copyright © 2017 Elsevier Inc. All rights reserved.

The ciliogenic transcription factor Rfx3 is required for the formation of the thalamocortical tract by regulating the patterning of prethalamus and ventral telencephalon.

PubMed

Magnani, Dario; Morlé, Laurette; Hasenpusch-Theil, Kerstin; Paschaki, Marie; Jacoby, Monique; Schurmans, Stéphane; Durand, Bénédicte; Theil, Thomas

2015-05-01

Primary cilia are complex subcellular structures that play key roles during embryogenesis by controlling the cellular response to several signaling pathways. Defects in the function and/or structure of primary cilia underlie a large number of human syndromes collectively referred to as ciliopathies. Often, ciliopathies are associated with mental retardation (MR) and malformation of the corpus callosum. However, the possibility of defects in other forebrain axon tracts, which could contribute to the cognitive disorders of these patients, has not been explored. Here, we investigate the formation of the corticothalamic/thalamocortical tracts in mice mutant for Rfx3, which regulates the expression of many genes involved in ciliogenesis and cilia function. Using DiI axon tracing and immunohistochemistry experiments, we show that some Rfx3(-/-) corticothalamic axons abnormally migrate toward the pial surface of the ventral telencephalon (VT). Some thalamocortical axons (TCAs) also fail to leave the diencephalon or abnormally project toward the amygdala. Moreover, the Rfx3(-/-) VT displays heterotopias containing attractive guidance cues and expressing the guidance molecules Slit1 and Netrin1. Finally, the abnormal projection of TCAs toward the amygdala is also present in mice carrying a mutation in the Inpp5e gene, which is mutated in Joubert Syndrome and which controls cilia signaling and stability. The presence of identical thalamocortical malformations in two independent ciliary mutants indicates a novel role for primary cilia in the formation of the corticothalamic/thalamocortical tracts by establishing the correct cellular environment necessary for its development. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

What is the function of centrioles?

PubMed

Marshall, Wallace F

2007-03-01

The function of centrioles has been controversial and remains incompletely resolved. This is because centrioles, in and of themselves, do not directly perform any physiological activity. Instead, their role is only to act as a jig or breadboard onto which other functional structures can be built. Centrioles are primarily involved in forming two structures-centrosomes and cilia. Centrioles bias the position of spindle pole formation, but because spindle poles can self-organize, the function of the centriole in mitosis is not obligatory. Consequently, lack of centrioles does not generally prevent mitosis, although recent experiments suggest acentriolar spindles have reduced fidelity of chromosome segregation. In contrast, centrioles are absolutely required for the assembly of cilia, including primary cilia that act as cellular antennae. Consistent with this requirement, it is now becoming clear that many ciliary diseases, including nephronophthisis, Bardet-Biedl syndrome, Meckel Syndrome, and Oral-Facial-Digital syndrome, are caused by defects in centriole-associated proteins.

Primary Cilia and Mammalian Hedgehog Signaling

PubMed Central

Bangs, Fiona; Anderson, Kathryn V.

2017-01-01

It has been a decade since it was discovered that primary cilia have an essential role in Hedgehog signaling in mammals. This discovery came from screens in the mouse that identified a set of genes that are required for both normal Hedgehog signaling and for the formation of primary cilia. Since then, dozens of mouse mutations have been identified that disrupt cilia in a variety of ways and have complex effects on Hedgehog signaling. Here we summarize the genetic and developmental studies used to deduce how Hedgehog signal transduction is linked to cilia and the complex effects that perturbation of cilia structure can have on Hh signaling. We conclude by describing the current status of our understanding of the cell-type specific regulation of ciliogenesis and how that determines the ability of cells to respond to Hedgehog ligands. PMID:27881449

Sperm-Associated Antigen–17 Gene Is Essential for Motile Cilia Function and Neonatal Survival

PubMed Central

Teves, Maria Eugenia; Zhang, Zhibing; Costanzo, Richard M.; Henderson, Scott C.; Corwin, Frank D.; Zweit, Jamal; Sundaresan, Gobalakrishnan; Subler, Mark; Salloum, Fadi N.; Rubin, Bruce K.

2013-01-01

Primary ciliary dyskinesia (PCD), resulting from defects in cilia assembly or motility, is caused by mutations in a number of genes encoding axonemal proteins. PCD phenotypes are variable, and include recurrent respiratory tract infections, bronchiectasis, hydrocephaly, situs inversus, and male infertility. We generated knockout mice for the sperm-associated antigen–17 (Spag17) gene, which encodes a central pair (CP) protein present in the axonemes of cells with “9 + 2” motile cilia or flagella. The targeting of Spag17 resulted in a severe phenotype characterized by immotile nasal and tracheal cilia, reduced clearance of nasal mucus, profound respiratory distress associated with lung fluid accumulation and disruption of the alveolar epithelium, cerebral ventricular expansion consistent with emerging hydrocephalus, failure to suckle, and neonatal demise within 12 hours of birth. Ultrastructural analysis revealed the loss of one CP microtubule in approximately one quarter of tracheal cilia axonemes, an absence of a C1 microtubule projection, and other less frequent CP structural abnormalities. SPAG6 and SPAG16 (CP proteins that interact with SPAG17) were increased in tracheal tissue from SPAG17-deficient mice. We conclude that Spag17 plays a critical role in the function and structure of motile cilia, and that neonatal lethality is likely explained by impaired airway mucociliary clearance. PMID:23418344

An Experimental and Computational Analysis of Primary Cilia Deflection Under Fluid Flow

PubMed Central

Downs, Matthew E.; Nguyen, An M.; Herzog, Florian A.; Hoey, David A.; Jacobs, Christopher R.

2013-01-01

In this work we have developed a novel model of the deflection of primary cilia experiencing fluid flow accounting for phenomena not previously considered. Specifically, we developed a large rotation formulation that accounts for rotation at the base of the cilium, the initial shape of the cilium and fluid drag at high deflection angles. We utilized this model to analyze full three dimensional datasets of primary cilia deflecting under fluid flow acquired with high-speed confocal microscopy. We found a wide variety of previously unreported bending shapes and behaviors. We also analyzed post-flow relaxation patterns. Results from our combined experimental and theoretical approach suggest that the average flexural rigidity of primary cilia might be higher than previously reported (Schwartz et al. 1997). In addition our findings indicate the mechanics of primary cilia are richly varied and mechanisms may exist to alter their mechanical behavior. PMID:22452422

Maintaining protein composition in cilia.

PubMed

Stephen, Louise A; Elmaghloob, Yasmin; Ismail, Shehab

2017-12-20

The primary cilium is a sensory organelle that is vital in regulating several signalling pathways. Unlike most organelles cilia are open to the rest of the cell, not enclosed by membranes. The distinct protein composition is crucial to the function of cilia and many signalling proteins and receptors are specifically concentrated within distinct compartments. To maintain this composition, a mechanism is required to deliver proteins to the cilium whilst another must counter the entropic tendency of proteins to distribute throughout the cell. The combination of the two mechanisms should result in the concentration of ciliary proteins to the cilium. In this review we will look at different cellular mechanisms that play a role in maintaining the distinct composition of cilia, including regulation of ciliary access and trafficking of ciliary proteins to, from and within the cilium.

Identification of G Protein-Coupled Receptors (GPCRs) in Primary Cilia and Their Possible Involvement in Body Weight Control.

PubMed

Omori, Yoshihiro; Chaya, Taro; Yoshida, Satoyo; Irie, Shoichi; Tsujii, Toshinori; Furukawa, Takahisa

2015-01-01

Primary cilia are sensory organelles that harbor various receptors such as G protein-coupled receptors (GPCRs). We analyzed subcellular localization of 138 non-odorant GPCRs. We transfected GPCR expression vectors into NIH3T3 cells, induced ciliogenesis by serum starvation, and observed subcellular localization of GPCRs by immunofluorescent staining. We found that several GPCRs whose ligands are involved in feeding behavior, including prolactin-releasing hormone receptor (PRLHR), neuropeptide FF receptor 1 (NPFFR1), and neuromedin U receptor 1 (NMUR1), localized to the primary cilia. In addition, we found that a short form of dopamine receptor D2 (DRD2S) is efficiently transported to the primary cilia, while a long form of dopamine receptor D2 (DRD2L) is rarely transported to the primary cilia. Using an anti-Prlhr antibody, we found that Prlhr localized to the cilia on the surface of the third ventricle in the vicinity of the hypothalamic periventricular nucleus. We generated the Npy2r-Cre transgenic mouse line in which Cre-recombinase is expressed under the control of the promoter of Npy2r encoding a ciliary GPCR. By mating Npy2r-Cre mice with Ift80 flox mice, we generated Ift80 conditional knockout (CKO) mice in which Npy2r-positive cilia were diminished in number. We found that Ift80 CKO mice exhibited a body weight increase. Our results suggest that Npy2r-positive cilia are important for body weight control.

Human basal body basics.

PubMed

Vertii, Anastassiia; Hung, Hui-Fang; Hehnly, Heidi; Doxsey, Stephen

2016-01-01

In human cells, the basal body (BB) core comprises a ninefold microtubule-triplet cylindrical structure. Distal and subdistal appendages are located at the distal end of BB, where they play indispensable roles in cilium formation and function. Most cells that arrest in the G0 stage of the cell cycle initiate BB docking at the plasma membrane followed by BB-mediated growth of a solitary primary cilium, a structure required for sensing the extracellular environment and cell signaling. In addition to the primary cilium, motile cilia are present in specialized cells, such as sperm and airway epithelium. Mutations that affect BB function result in cilia dysfunction. This can generate syndromic disorders, collectively called ciliopathies, for which there are no effective treatments. In this review, we focus on the features and functions of BBs and centrosomes in Homo sapiens.

Quantifying Ciliary Dynamics during Assembly Reveals Step-wise Waveform Maturation in Airway Cells.

PubMed

Oltean, Alina; Schaffer, Andrew J; Bayly, Philip V; Brody, Steven L

2018-05-31

Motile cilia are essential for clearance of particulates and pathogens from airways. For effective transport, ciliary motor proteins and axonemal structures interact to generate the rhythmic, propulsive bending, but the mechanisms that produce a dynamic waveform remain incompletely understood. Biomechanical measures of human cilia motion and their relationships to cilia assembly are needed to illuminate the biophysics of normal cilia function, and to quantify dysfunction in ciliopathies. To these ends, we analyzed cilia motion from high-speed video microscopy of ciliated cells sampled from human lung airways compared to primary-culture cells that undergo ciliogenesis in vitro. Quantitative assessment of waveform parameters showed variations in waveform shape between individual cilia; however, general trends in waveform parameters emerged, associated with progression of cilia length and stage of differentiation. When cilia emerged from cultured cells, beat frequency was initially elevated, then fell and remained stable as cilia lengthened. In contrast, the average bending amplitude and the ability to generate force gradually increased and eventually approached values observed in ex vivo samples. Dynein arm motor proteins DNAH5, DNAH9, DNAH11, and DNAH6 were localized within specific regions of the axoneme in the ex vivo cells; however distinct stages of in vitro waveform development identified by biomechanical features were associated with the progressive movement of dyneins to the appropriate proximal or distal sections of the cilium. These observations suggest that the step-wise variation in waveform development during ciliogenesis is dependent on cilia length and potentially outer dynein arm assembly.

Defective ciliogenesis in thyroid hürthle cell tumors is associated with increased autophagy

PubMed Central

Lee, Junguee; Yi, Shinae; Kang, Yea Eun; Chang, Joon Young; Kim, Jung Tae; Sul, Hae Joung; Kim, Jong Ok; Kim, Jin Man; Kim, Joon; Porcelli, Anna Maria; Kim, Koon Soon; Shong, Minho

2016-01-01

Primary cilia are found in the apical membrane of thyrocytes, where they may play a role in the maintenance of follicular homeostasis. In this study, we examined the distribution of primary cilia in the human thyroid cancer to address the involvement of abnormal ciliogenesis in different thyroid cancers. We examined 92 human thyroid tissues, including nodular hyperplasia, Hashimoto's thyroiditis, follicular tumor, Hürthle cell tumor, and papillary carcinoma to observe the distribution of primary cilia. The distribution and length of primary cilia facing the follicular lumen were uniform across variable-sized follicles in the normal thyroid gland. However, most Hürthle cells found in benign and malignant thyroid diseases were devoid of primary cilia. Conventional variant of papillary carcinoma (PTC) displayed longer primary cilia than those of healthy tissue, whereas both the frequency and length of primary cilia were decreased in oncocytic variant of PTC. In addition, ciliogenesis was markedly defective in primary Hürthle cell tumors, including Hürthle cell adenomas and carcinomas, which showed higher level of autophagosome biogenesis. Remarkably, inhibition of autophagosome formation by Atg5 silencing or treatment with pharmacological inhibitors of autophagosome formation restored ciliogenesis in the Hürthle cell carcinoma cell line XTC.UC1 which exhibits a high basal autophagic flux. Moreover, the inhibition of autophagy promoted the accumulation of two factors critical for ciliogenesis, IFT88 and ARL13B. These results suggest that abnormal ciliogenesis, a common feature of Hürthle cells in diseased thyroid glands, is associated with increased basal autophagy. PMID:27816963

Emerging ciliopathies: are respiratory cilia compromised in Usher syndrome?

PubMed

Piatti, G; De Santi, M M; Brogi, M; Castorina, P; Ambrosetti, U

2014-01-01

Usher syndrome is a ciliopathy involving photoreceptors and cochlear hair cells (sensory cilia): since sensory and motor ciliopathies can overlap, we analysed the respiratory cilia (motile) in 17 patients affected by Usher syndrome and 18 healthy control subject. We studied the mucociliary transport time with the saccharine test, ciliary motility and ultrastructure of respiratory cilia obtained by nasal brushing; we also recorded the classical respiratory function values by spirometry. All enrolled subjects showed normal respiratory function values. The mean mucociliary transport time with saccharine was 22.33 ± 17.96 min, which is in the range of normal values. The mean ciliary beat frequency of all subjects was 8.81 ± 2.18 Hz, which is a value approaching the lower physiological limit. None of the classical ciliary alterations characterizing the "ciliary primary dyskinesia" was detected, although two patients showed alterations in number and arrangement of peripheral microtubules and one patient had abnormal ciliary roots. Respiratory cilia in Usher patients don't seem to have evident ultrastructural alterations, as expected, but the fact that the ciliary motility appeared slightly reduced could emphasize that a rigid distinction between sensory and motor ciliopathies may not reflect what really occurs. Copyright © 2014 Elsevier Inc. All rights reserved.

Mechanical Properties of a Primary Cilium Measured by Resonant Oscillation

NASA Astrophysics Data System (ADS)

Resnick, Andrew

Primary cilia are ubiquitous mammalian cellular substructures implicated in an ever-increasing number of regulatory pathways. The well-established `ciliary hypothesis' states that physical bending of the cilium (for example, due to fluid flow) initiates signaling cascades, yet the mechanical properties of the cilium remain incompletely measured, resulting in confusion regarding the biological significance of flow-induced ciliary mechanotransduction. In this work we measure the mechanical properties of a primary cilium by using an optical trap to induce resonant oscillation of the structure. Our data indicate 1), the primary cilium is not a simple cantilevered beam, 2), the base of the cilium may be modeled as a nonlinear rotatory spring, the linear spring constant `k' of the cilium base calculated to be (4.6 +/- 0.62)*10-12 N/rad and nonlinear spring constant ` α' to be (-1 +/- 0.34) *10-10 N/rad2 , and 3) the ciliary base may be an essential regulator of mechanotransduction signalling. Our method is also particularly suited to measure mechanical properties of nodal cilia, stereocilia, and motile cilia, anatomically similar structures with very different physiological functions.

CCDC65 Mutation Causes Primary Ciliary Dyskinesia with Normal Ultrastructure and Hyperkinetic Cilia

PubMed Central

Horani, Amjad; Brody, Steven L.; Ferkol, Thomas W.; Shoseyov, David; Wasserman, Mollie G.; Ta-shma, Asaf; Wilson, Kate S.; Bayly, Philip V.; Amirav, Israel; Cohen-Cymberknoh, Malena; Dutcher, Susan K.; Elpeleg, Orly; Kerem, Eitan

2013-01-01

Background Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating. Methods Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion. Results A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells. Conclusion Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis. PMID:23991085

Ciliatoxicity in human primary bronchiolar epithelial cells after repeated exposure at the air-liquid interface with native mainstream smoke of K3R4F cigarettes with and without charcoal filter.

PubMed

Aufderheide, Michaela; Scheffler, Stefanie; Ito, Shigeaki; Ishikawa, Shinkichi; Emura, Makito

2015-01-01

Mucociliary clearance is the primary physical mechanism to protect the human airways against harmful effects of inhaled particles. Environmental factors play a significant role in the impairment of this defense mechanism, whereas cigarette smoke is discussed to be one of the clinically most important causes. Impaired mucociliary clearance in smokers has been connected to changes in ciliated cells such as decreased numbers, altered structure and beat frequency. Clinical studies have shown that cilia length is reduced in healthy smokers and that long-term exposure to cigarette smoke leads to reduced numbers of ciliated cells in mice. We present an in vitro model of primary normal human bronchiolar epithelial (NHBE) cells with in vivo like morphology to study the influence of cigarette mainstream smoke on ciliated cells. We exposed mucociliary differentiated cultures repeatedly to non-toxic concentrations of mainstream cigarette smoke (4 cigarettes, 5 days/week, 8 repetitions in total) at the air-liquid interface. Charcoal filter tipped cigarettes were compared to those being equipped with standard cellulose acetate filters. Histopathological analyses of the exposed cultures showed a reduction of cilia bearing cells, shortening of existing cilia and finally disappearance of all cilia in cigarette smoke exposed cells. In cultures exposed to charcoal filtered cigarette smoke, little changes in cilia length were seen after four exposure repetitions, but those effects were reversed after a two day recovery period. Those differences indicate that volatile organic compounds, being removed by the charcoal filter tip, affect primary bronchiolar epithelial cells concerning their cilia formation and function comparable with the in vivo situation. In conclusion, our in vitro model presents a valuable tool to study air-borne ciliatoxic compounds. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

Primary cilia are increased in number and demonstrate structural abnormalities in human cancer.

PubMed

Yasar, Binnaz; Linton, Kim; Slater, Christian; Byers, Richard

2017-07-01

Primary cilia play an important role in the regulation of cell signalling pathways and are thought to have a role in cancer but have seldom been studied in human cancer samples. Primary cilia were visualised by dual immunofluorescence for anti-CROCC (ciliary rootlet coiled-coil) and anti-tubulin in a range of human cancers (including carcinomas of stomach, pancreas, prostate, lung and colon, lobular and ductal breast cancers and follicular lymphoma) and in matched normal tissue (stomach, pancreas, lung, large and small intestines, breast and reactive lymph nodes) samples using a tissue microarray; their frequency, association with proliferation, was measured by Ki-67 staining and their structure was analysed. Compared with normal tissues, primary cilia frequency was significantly elevated in adenocarcinoma of the lung (2.75% vs 1.85%, p=0.016), adenocarcinoma of the colon (3.80% vs 2.43%, respectively, p=0.017), follicular lymphoma (1.18% vs 0.83%, p=0.003) and pancreatic adenocarcinoma (7.00% vs 5.26%, p=0.002); there was no statistically significant difference compared with normal control tissue for gastric and prostatic adenocarcinomas or for lobular and ductal breast cancers. Additionally, structural abnormalities of primary cilia were identified in cancer tissues, including elongation of the axoneme, multiple basal bodies and branching of the axoneme. Ki-67 scores ranged from 0.7% to 78.4% and showed no statistically significant correlation with primary cilia frequency across all tissues (p=0.1501). The results show upregulation of primary cilia and the presence of structural defects in a wide range of human cancer tissue samples demonstrating association of dysregulation of primary cilia with human cancer. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Identification of G Protein-Coupled Receptors (GPCRs) in Primary Cilia and Their Possible Involvement in Body Weight Control

PubMed Central

Omori, Yoshihiro; Chaya, Taro; Yoshida, Satoyo; Irie, Shoichi; Tsujii, Toshinori; Furukawa, Takahisa

2015-01-01

Primary cilia are sensory organelles that harbor various receptors such as G protein-coupled receptors (GPCRs). We analyzed subcellular localization of 138 non-odorant GPCRs. We transfected GPCR expression vectors into NIH3T3 cells, induced ciliogenesis by serum starvation, and observed subcellular localization of GPCRs by immunofluorescent staining. We found that several GPCRs whose ligands are involved in feeding behavior, including prolactin-releasing hormone receptor (PRLHR), neuropeptide FF receptor 1 (NPFFR1), and neuromedin U receptor 1 (NMUR1), localized to the primary cilia. In addition, we found that a short form of dopamine receptor D2 (DRD2S) is efficiently transported to the primary cilia, while a long form of dopamine receptor D2 (DRD2L) is rarely transported to the primary cilia. Using an anti-Prlhr antibody, we found that Prlhr localized to the cilia on the surface of the third ventricle in the vicinity of the hypothalamic periventricular nucleus. We generated the Npy2r-Cre transgenic mouse line in which Cre-recombinase is expressed under the control of the promoter of Npy2r encoding a ciliary GPCR. By mating Npy2r-Cre mice with Ift80 flox mice, we generated Ift80 conditional knockout (CKO) mice in which Npy2r-positive cilia were diminished in number. We found that Ift80 CKO mice exhibited a body weight increase. Our results suggest that Npy2r-positive cilia are important for body weight control. PMID:26053317

Mechanosensory Genes Pkd1 and Pkd2 Contribute to the Planar Polarization of Brain Ventricular Epithelium

PubMed Central

Herranz-Pérez, Vicente; Nakatani, Jin; Boletta, Alessandra; García-Verdugo, José Manuel

2015-01-01

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. PMID:26245976

Loss of SPEF2 Function in Mice Results in Spermatogenesis Defects and Primary Ciliary Dyskinesia1

PubMed Central

Sironen, Anu; Kotaja, Noora; Mulhern, Howard; Wyatt, Todd A.; Sisson, Joseph H.; Pavlik, Jacqueline A.; Miiluniemi, Mari; Fleming, Mark D.; Lee, Lance

2011-01-01

Primary ciliary dyskinesia (PCD) results from defects in motile cilia function. Mice homozygous for the mutation big giant head (bgh) have several abnormalities commonly associated with PCD, including hydrocephalus, male infertility, and sinusitis. In the present study, we use a variety of histopathological and cell biological techniques to characterize the bgh phenotype, and we identify the bgh mutation using a positional cloning approach. Histopathological, immunofluorescence, and electron microscopic analyses demonstrate that the male infertility results from shortened flagella and disorganized axonemal and accessory structures in elongating spermatids and mature sperm. In addition, there is a reduced number of elongating spermatids during spermatogenesis and mature sperm in the epididymis. Histological analyses show that the hydrocephalus is characterized by severe dilatation of the lateral ventricles and that bgh sinuses have an accumulation of mucus infiltrated by neutrophils. In contrast to the sperm phenotype, electron microscopy demonstrates that mutant respiratory epithelial cilia are ultrastructurally normal, but video microscopic analysis shows that their beat frequency is lower than that of wild-type cilia. Through a positional cloning approach, we identified two sequence variants in the gene encoding sperm flagellar protein 2 (SPEF2), which has been postulated to play an important role in spermatogenesis and flagellar assembly. A causative nonsense mutation was validated by Western blot analysis, strongly suggesting that the bgh phenotype results from the loss of SPEF2 function. Taken together, the data in this study demonstrate that SPEF2 is required for cilia function and identify a new genetic cause of PCD in mice. PMID:21715716

Primary Cilia Modulate IHH Signal Transduction in Response to Hydrostatic Loading of Growth Plate Chondrocytes

PubMed Central

Shao, Y, Yvonne Y.; Wang, Lai; Welter, J, Jean F.; Ballock, R. Tracy

2011-01-01

Indian Hedgehog (Ihh) is a key component of the regulatory apparatus governing chondrocyte proliferation and differentiation in the growth plate. Recent studies have demonstrated that the primary cilium is the site of Ihh signaling within the cell, and that primary cilia are essential for bone and cartilage formation. Primary cilia are also postulated to act as mechanosensory organelles that transduce mechanical forces acting on the cell into biological signals. In this study, we used a hydrostatic compression system to examine Ihh signal transduction under the influence of mechanical load. Our results demonstrate that hydrostatic compression increased both Ihh gene expression and Ihh-responsive Gli-luciferase activity. These increases were aborted by disrupting the primary cilia structure with chloral hydrate. These results suggest that growth plate chondrocytes respond to hydrostatic loading by increasing Ihh signaling, and that the primary cilium is required for this mechano-biological signal transduction to occur. PMID:21930256

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jumat, Muhammad Raihan; Yan, Yan; Ravi, Laxmi Iyer

The distribution of cilia and the respiratory syncytial virus (RSV) nucleocapsid (N) protein, fusion (F) protein, attachment (G) protein, and M2-1 protein in human ciliated nasal epithelial cells was examined at between 1 and 5 days post-infection (dpi). All virus structural proteins were localized at cell surface projections that were distinct from cilia. The F protein was also trafficked into the cilia, and while its presence increased as the infection proceeded, the N protein was not detected in the cilia at any time of infection. The presence of the F protein in the cilia correlated with cellular changes in themore » cilia and reduced cilia function. At 5 dpi extensive cilia loss and further reduced cilia function was noted. These data suggested that although RSV morphogenesis occurs at non-cilia locations on ciliated nasal epithelial cells, RSV infection induces changes in the cilia body that leads to extensive cilia loss. - Highlights: • Respiratory syncytial virus (RSV) infects nasal ciliated epithelial cells. • Virus morphogenesis occurs within filamentous projections distinct from cilia. • The RSV N protein was not detected in the cilia at any time during infection. • Trafficking of the F protein into the cilia occurred early in infection. • Presence of the F protein in cilia correlated with impaired cilia function.« less

Asymmetric Distribution of Primary Cilia Allocates Satellite Cells for Self-Renewal.

PubMed

Jaafar Marican, Nur Hayati; Cruz-Migoni, Sara B; Borycki, Anne-Gaëlle

2016-06-14

Regeneration of vertebrate skeletal muscles requires satellite cells, a population of stem cells that are quiescent in normal conditions and divide, differentiate, and self-renew upon activation triggered by exercise, injury, and degenerative diseases. Satellite cell self-renewal is essential for long-term tissue homeostasis, and previous work has identified a number of external cues that control this process. However, little is known of the possible intrinsic control mechanisms of satellite cell self-renewal. Here, we show that quiescent satellite cells harbor a primary cilium, which is rapidly disassembled upon entry into the cell cycle. Contrasting with a commonly accepted belief, cilia reassembly does not occur uniformly in cells exiting the cell cycle. We found that primary cilia reassemble preferentially in cells committed to self-renew, and disruption of cilia reassembly causes a specific deficit in self-renewing satellite cells. These observations indicate that primary cilia provide an intrinsic cue essential for satellite cell self-renewal. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Topography of calcium phosphate ceramics regulates primary cilia length and TGF receptor recruitment associated with osteogenesis.

PubMed

Zhang, Jingwei; Dalbay, Melis T; Luo, Xiaoman; Vrij, Erik; Barbieri, Davide; Moroni, Lorenzo; de Bruijn, Joost D; van Blitterswijk, Clemens A; Chapple, J Paul; Knight, Martin M; Yuan, Huipin

2017-07-15

The surface topography of synthetic biomaterials is known to play a role in material-driven osteogenesis. Recent studies show that TGFβ signalling also initiates osteogenic differentiation. TGFβ signalling requires the recruitment of TGFβ receptors (TGFβR) to the primary cilia. In this study, we hypothesize that the surface topography of calcium phosphate ceramics regulates stem cell morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation. We developed a 2D system using two types of tricalcium phosphate (TCP) ceramic discs with identical chemistry. One sample had a surface topography at micron-scale (TCP-B, with a bigger surface structure dimension) whilst the other had a surface topography at submicron scale (TCP-S, with a smaller surface structure dimension). In the absence of osteogenic differentiation factors, human bone marrow stromal cells (hBMSCs) were more spread on TCP-S than on TCP-B with alterations in actin organization and increased primary cilia prevalence and length. The cilia elongation on TCP-S was similar to that observed on glass in the presence of osteogenic media and was followed by recruitment of transforming growth factor-β RII (p-TGFβ RII) to the cilia axoneme. This was associated with enhanced osteogenic differentiation of hBMSCs on TCP-S, as shown by alkaline phosphatase activity and gene expression for key osteogenic markers in the absence of additional osteogenic growth factors. Similarly, in vivo after a 12-week intramuscular implantation in dogs, TCP-S induced bone formation while TCP-B did not. It is most likely that the surface topography of calcium phosphate ceramics regulates primary cilia length and ciliary recruitment of p-TGFβ RII associated with osteogenesis and bone formation. This bioengineering control of osteogenesis via primary cilia modulation may represent a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery applications. The surface topography of synthetic biomaterials plays important roles in material-driven osteogenesis. The data presented herein have shown that the surface topography of calcium phosphate ceramics regulates mesenchymal stromal cells (e.g., human bone marrow mesenchymal stromal cells, hBMSCs) with respect to morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation in vitro. Together with bone formation in vivo, our results suggested a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery by the bioengineering control of osteogenesis via primary cilia modulation. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

[Non-ciliary functions of cilia proteins].

PubMed

Taulet, Nicolas; Delaval, Bénédicte

2014-11-01

Cilia proteins have long been characterized for their role in cilia formation and function, and their implications in ciliopathies. However, several cellular defects induced by cilia proteins deregulation suggest that they could have non-ciliary roles. Indeed, several non-ciliary functions have been recently characterized for cilia proteins including roles in intra-cellular and in vesicular transport, in spindle orientation or in the maintenance of genomic stability. These observations thus raise the crucial question of the contribution of non-ciliary functions of cilia proteins to the pathological manifestations associated with ciliopathies such as polycystic kidney disease. © 2014 médecine/sciences – Inserm.

Motile cilia of human airway epithelia contain hedgehog signaling components that mediate noncanonical hedgehog signaling.

PubMed

Mao, Suifang; Shah, Alok S; Moninger, Thomas O; Ostedgaard, Lynda S; Lu, Lin; Tang, Xiao Xiao; Thornell, Ian M; Reznikov, Leah R; Ernst, Sarah E; Karp, Philip H; Tan, Ping; Keshavjee, Shaf; Abou Alaiwa, Mahmoud H; Welsh, Michael J

2018-02-06

Differentiated airway epithelia produce sonic hedgehog (SHH), which is found in the thin layer of liquid covering the airway surface. Although previous studies showed that vertebrate HH signaling requires primary cilia, as airway epithelia mature, the cells lose primary cilia and produce hundreds of motile cilia. Thus, whether airway epithelia have apical receptors for SHH has remained unknown. We discovered that motile cilia on airway epithelial cells have HH signaling proteins, including patched and smoothened. These cilia also have proteins affecting cAMP-dependent signaling, including Gα i and adenylyl cyclase 5/6. Apical SHH decreases intracellular levels of cAMP, which reduces ciliary beat frequency and pH in airway surface liquid. These results suggest that apical SHH may mediate noncanonical HH signaling through motile cilia to dampen respiratory defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses. Copyright © 2018 the Author(s). Published by PNAS.

A truncating mutation of Alms1 reduces the number of hypothalamic neuronal cilia in obese mice.

PubMed

Heydet, Déborah; Chen, Lesley X; Larter, Claire Z; Inglis, Chrystal; Silverman, Michael A; Farrell, Geoffrey C; Leroux, Michel R

2013-01-01

Primary cilia are ubiquitous cellular antennae whose dysfunction collectively causes various disorders, including vision and hearing impairment, as well as renal, skeletal, and central nervous system anomalies. One ciliopathy, Alström syndrome, is closely related to Bardet-Biedl syndrome (BBS), sharing amongst other phenotypic features morbid obesity. As the cellular and molecular links between weight regulation and cilia are poorly understood, we used the obese mouse strain foz/foz, bearing a truncating mutation in the Alström syndrome protein (Alms1), to help elucidate why it develops hyperphagia, leading to early onset obesity and metabolic anomalies. Our in vivo studies reveal that Alms1 localizes at the base of cilia in hypothalamic neurons, which are implicated in the control of satiety. Alms1 is lost from this location in foz/foz mice, coinciding with a strong postnatal reduction (∼70%) in neurons displaying cilia marked with adenylyl cyclase 3 (AC3), a signaling protein implicated in obesity. Notably, the reduction in AC3-bearing cilia parallels the decrease in cilia containing two appetite-regulating proteins, Mchr1 and Sstr3, as well as another established Arl13b ciliary marker, consistent with progressive loss of cilia during development. Together, our results suggest that Alms1 maintains the function of neuronal cilia implicated in weight regulation by influencing the maintenance and/or stability of the organelle. Given that Mchr1 and Sstr3 localization to remaining cilia is maintained in foz/foz animals but known to be lost from BBS knockout mice, our findings suggest different molecular etiologies for the satiety defects associated with the Alström syndrome and BBS ciliopathies. Copyright © 2012 Wiley Periodicals, Inc.

Non-essential role for cilia in coordinating precise alignment of lens fibres

PubMed Central

Sugiyama, Yuki; Shelley, Elizabeth J.; Yoder, Bradley K.; Kozmik, Zbynek; May-Simera, Helen L.; Beales, Philip L.; Lovicu, Frank J.; McAvoy, John W.

2016-01-01

The primary cilium, a microtubule-based organelle found in most cells, is a centre for mechano-sensing fluid movement and cellular signalling, notably through the Hedgehog pathway. We recently found that each lens fibre cell has an apically situated primary cilium that is polarised to the side of the cell facing the anterior pole of the lens. The direction of polarity is similar in neighbouring cells so that in the global view, lens fibres exhibit planar cell polarity (PCP) along the equatorial-anterior polar axis. Ciliogenesis has been associated with the establishment of PCP, although the exact relationship between PCP and the role of cilia is still controversial. To test the hypothesis that the primary cilia have a role in coordinating the precise alignment/orientation of the fibre cells, IFT88, a key component of the intraflagellar transport (IFT) complex, was removed specifically from the lens at different developmental stages using several lens-specific Cre-expressing mouse lines (MLR10- and LR-Cre). Irrespective of which Cre-line was adopted, both demonstrated that in IFT88-depleted cells, the ciliary axoneme was absent or substantially shortened, confirming the disruption of primary cilia formation. However no obvious histological defects were detected even when IFT88 was removed from the lens placode as early as E9.5. Specifically, the lens fibres aligned/oriented towards the poles to form the characteristic Y-shaped sutures as normal. Consistent with this, in primary lens epithelial explants prepared from these conditional knockout mouse lenses, the basal bodies still showed polarised localisation at the apical surface of elongating cells upon FGF-induced fibre differentiation. We further investigated the lens phenotype in knockouts of Bardet–Biedl Syndrome (BBS) proteins 4 and 8, the components of the BBSome complex which modulate ciliary function. In these BBS4 and 8 knockout lenses, again we found the pattern of the anterior sutures formed by the apical tips of elongating/migrating fibres were comparable to the control lenses. Taken together, these results indicate that primary cilia do not play an essential role in the precise cellular alignment/orientation of fibre cells. Thus, it appears that in the lens cilia are not required to establish PCP. PMID:26825015

Traumatic Brain Injury-Induced Ependymal Ciliary Loss Decreases Cerebral Spinal Fluid Flow

PubMed Central

Xiong, Guoxiang; Elkind, Jaclynn A.; Kundu, Suhali; Smith, Colin J.; Antunes, Marcelo B.; Tamashiro, Edwin; Kofonow, Jennifer M.; Mitala, Christina. M.; Stein, Sherman C.; Grady, M. Sean; Einhorn, Eugene; Cohen, Noam A.

2014-01-01

Abstract Traumatic brain injury (TBI) afflicts up to 2 million people annually in the United States and is the primary cause of death and disability in young adults and children. Previous TBI studies have focused predominantly on the morphological, biochemical, and functional alterations of gray matter structures, such as the hippocampus. However, little attention has been given to the brain ventricular system, despite the fact that altered ventricular function is known to occur in brain pathologies. In the present study, we investigated anatomical and functional alterations to mouse ventricular cilia that result from mild TBI. We demonstrate that TBI causes a dramatic decrease in cilia. Further, using a particle tracking technique, we demonstrate that cerebrospinal fluid flow is diminished, thus potentially negatively affecting waste and nutrient exchange. Interestingly, injury-induced ventricular system pathology resolves completely by 30 days after injury as ependymal cell ciliogenesis restores cilia density to uninjured levels in the affected lateral ventricle. PMID:24749541

Transmembrane protein OSTA-1 shapes sensory cilia morphology via regulation of intracellular membrane trafficking in C. elegans.

PubMed

Olivier-Mason, Anique; Wojtyniak, Martin; Bowie, Rachel V; Nechipurenko, Inna V; Blacque, Oliver E; Sengupta, Piali

2013-04-01

The structure and function of primary cilia are critically dependent on intracellular trafficking pathways that transport ciliary membrane and protein components. The mechanisms by which these trafficking pathways are regulated are not fully characterized. Here we identify the transmembrane protein OSTA-1 as a new regulator of the trafficking pathways that shape the morphology and protein composition of sensory cilia in C. elegans. osta-1 encodes an organic solute transporter alpha-like protein, mammalian homologs of which have been implicated in membrane trafficking and solute transport, although a role in regulating cilia structure has not previously been demonstrated. We show that mutations in osta-1 result in altered ciliary membrane volume, branch length and complexity, as well as defects in localization of a subset of ciliary transmembrane proteins in different sensory cilia types. OSTA-1 is associated with transport vesicles, localizes to a ciliary compartment shown to house trafficking proteins, and regulates both retrograde and anterograde flux of the endosome-associated RAB-5 small GTPase. Genetic epistasis experiments with sensory signaling, exocytic and endocytic proteins further implicate OSTA-1 as a crucial regulator of ciliary architecture via regulation of cilia-destined trafficking. Our findings suggest that regulation of transport pathways in a cell type-specific manner contributes to diversity in sensory cilia structure and might allow dynamic remodeling of ciliary architecture via multiple inputs.

Primary cilia modulate Ihh signal transduction in response to hydrostatic loading of growth plate chondrocytes.

PubMed

Shao, Yvonne Y; Wang, Lai; Welter, Jean F; Ballock, R Tracy

2012-01-01

Indian hedgehog (Ihh) is a key component of the regulatory apparatus governing chondrocyte proliferation and differentiation in the growth plate. Recent studies have demonstrated that the primary cilium is the site of Ihh signaling within the cell, and that primary cilia are essential for bone and cartilage formation. Primary cilia are also postulated to act as mechanosensory organelles that transduce mechanical forces acting on the cell into biological signals. In this study, we used a hydrostatic compression system to examine Ihh signal transduction under the influence of mechanical load. Our results demonstrate that hydrostatic compression increased both Ihh gene expression and Ihh-responsive Gli-luciferase activity. These increases were aborted by disrupting the primary cilia structure with chloral hydrate. These results suggest that growth plate chondrocytes respond to hydrostatic loading by increasing Ihh signaling, and that the primary cilium is required for this mechano-biological signal transduction to occur. Copyright © 2011 Elsevier Inc. All rights reserved.

Cilia distribution and polarity in the epithelial lining of the mouse middle ear cavity

PubMed Central

Luo, Wenwei; Yi, Hong; Taylor, Jeannette; Li, Jian-dong; Chi, Fanglu; Todd, N. Wendell; Lin, Xi; Ren, Dongdong; Chen, Ping

2017-01-01

The middle ear conducts sound to the cochlea for hearing. Otitis media (OM) is the most common illness in childhood. Moreover, chronic OM with effusion (COME) is the leading cause of conductive hearing loss. Clinically, COME is highly associated with Primary Ciliary Dyskinesia, implicating significant contributions of cilia dysfunction to COME. The understanding of middle ear cilia properties that are critical to OM susceptibility, however, is limited. Here, we confirmed the presence of a ciliated region near the Eustachian tube orifice at the ventral region of the middle ear cavity, consisting mostly of a lumen layer of multi-ciliated and a layer of Keratin-5-positive basal cells. We also found that the motile cilia are polarized coordinately and display a planar cell polarity. Surprisingly, we also found a region of multi-ciliated cells that line the posterior dorsal pole of the middle ear cavity which was previously thought to contain only non-ciliated cells. Our study provided a more complete understanding of cilia distribution and revealed for the first time coordinated polarity of cilia in the epithelium of the mammalian middle ear, thus illustrating novel structural features that are likely critical for middle ear functions and related to OM susceptibility. PMID:28358397

Lack of centrioles and primary cilia in STIL−/− mouse embryos

PubMed Central

David, Ahuvit; Liu, Fengying; Tibelius, Alexandra; Vulprecht, Julia; Wald, Diana; Rothermel, Ulrike; Ohana, Reut; Seitel, Alexander; Metzger, Jasmin; Ashery-Padan, Ruth; Meinzer, Hans-Peter; Gröne, Hermann-Josef; Izraeli, Shai; Krämer, Alwin

2014-01-01

Although most animal cells contain centrosomes, consisting of a pair of centrioles, their precise contribution to cell division and embryonic development is unclear. Genetic ablation of STIL, an essential component of the centriole replication machinery in mammalian cells, causes embryonic lethality in mice around mid gestation associated with defective Hedgehog signaling. Here, we describe, by focused ion beam scanning electron microscopy, that STIL−/− mouse embryos do not contain centrioles or primary cilia, suggesting that these organelles are not essential for mammalian development until mid gestation. We further show that the lack of primary cilia explains the absence of Hedgehog signaling in STIL−/− cells. Exogenous re-expression of STIL or STIL microcephaly mutants compatible with human survival, induced non-templated, de novo generation of centrioles in STIL−/− cells. Thus, while the abscence of centrioles is compatible with mammalian gastrulation, lack of centrioles and primary cilia impairs Hedgehog signaling and further embryonic development. PMID:25486474

Lack of centrioles and primary cilia in STIL(-/-) mouse embryos.

PubMed

David, Ahuvit; Liu, Fengying; Tibelius, Alexandra; Vulprecht, Julia; Wald, Diana; Rothermel, Ulrike; Ohana, Reut; Seitel, Alexander; Metzger, Jasmin; Ashery-Padan, Ruth; Meinzer, Hans-Peter; Gröne, Hermann-Josef; Izraeli, Shai; Krämer, Alwin

2014-01-01

Although most animal cells contain centrosomes, consisting of a pair of centrioles, their precise contribution to cell division and embryonic development is unclear. Genetic ablation of STIL, an essential component of the centriole replication machinery in mammalian cells, causes embryonic lethality in mice around mid gestation associated with defective Hedgehog signaling. Here, we describe, by focused ion beam scanning electron microscopy, that STIL(-/-) mouse embryos do not contain centrioles or primary cilia, suggesting that these organelles are not essential for mammalian development until mid gestation. We further show that the lack of primary cilia explains the absence of Hedgehog signaling in STIL(-/-) cells. Exogenous re-expression of STIL or STIL microcephaly mutants compatible with human survival, induced non-templated, de novo generation of centrioles in STIL(-/-) cells. Thus, while the abscence of centrioles is compatible with mammalian gastrulation, lack of centrioles and primary cilia impairs Hedgehog signaling and further embryonic development.

Reduction of the immunostainable length of the hippocampal dentate granule cells' primary cilia in 3xAD-transgenic mice producing human A{beta}{sub 1-42} and tau

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakravarthy, Balu, E-mail: Balu.Chakravarthy@nrc-cnrc.gc.ca; Gaudet, Chantal; Menard, Michel

2012-10-12

Highlights: Black-Right-Pointing-Pointer A{beta} and tau-induced neurofibrillary tangles play a key role in Alzheimer's disease. Black-Right-Pointing-Pointer A{beta}{sub 1-42} and mutant tau protein together reduce the primary cilium length. Black-Right-Pointing-Pointer This shortening likely reduces cilium-dependent neurogenesis and memory function. Black-Right-Pointing-Pointer This provides a model of an A{beta}/tau targeting of a neuronal signaling organelle. -- Abstract: The hippocampal dentate gyrus is one of the two sites of continuous neurogenesis in adult rodents and humans. Virtually all dentate granule cells have a single immobile cilium with a microtubule spine or axoneme covered with a specialized cell membrane loaded with receptors such as the somatostatinmore » receptor 3 (SSTR3), and the p75 neurotrophin receptor (p75{sup NTR}). The signals from these receptors have been reported to stimulate neuroprogenitor proliferation and the post-mitotic maturation of newborn granule cells into functioning granule cells. We have found that in 6-24-months-old triple transgenic Alzheimer's disease model mice (3xTg-AD) producing both A{beta}{sub 1-42} and the mutant human tau protein tau{sub P301L,} the dentate granule cells still had immunostainable SSTR3- and p75{sup NTR}-bearing cilia but they were only half the length of the immunostained cilia in the corresponding wild-type mice. However, the immunostainable length of the granule cell cilia was not reduced either in 2xTg-AD mice accumulating large amounts of A{beta}{sub 1-42} or in mice accumulating only a mutant human tau protein. Thus it appears that a combination of A{beta}{sub 1-42} and tau protein accumulation affects the levels of functionally important receptors in 3xTg-AD mice. These observations raise the important possibility that structural and functional changes in granule cell cilia might have a role in AD.« less

Chronic Fluid Flow Is an Environmental Modifier of Renal Epithelial Function

PubMed Central

Resnick, Andrew

2011-01-01

Although solitary or sensory cilia are present in most cells of the body and their existence has been known since the sixties, very little is been known about their functions. One suspected function is fluid flow sensing- physical bending of cilia produces an influx of Ca++, which can then result in a variety of activated signaling pathways. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a progressive disease, typically appearing in the 5th decade of life and is one of the most common monogenetic inherited human diseases, affecting approximately 600,000 people in the United States. Because ADPKD is a slowly progressing disease, I asked how fluid flow may act, via the primary cilium, to alter epithelial physiology during the course of cell turnover. I performed an experiment to determine under what conditions fluid flow can result in a change of function of renal epithelial tissue. A wildtype epithelial cell line derived the cortical collecting duct of a heterozygous offspring of the Immortomouse (Charles River Laboratory) was selected as our model system. Gentle orbital shaking was used to induce physiologically relevant fluid flow, and periodic measurements of the transepithelial Sodium current were performed. At the conclusion of the experiment, mechanosensitive proteins of interest were visualized by immunostaining. I found that fluid flow, in itself, modifies the transepithelial sodium current, cell proliferation, and the actin cytoskeleton. These results significantly impact the understanding of both the mechanosensation function of primary cilia as well as the understanding of ADPKD disease progression. PMID:22046444

Intraflagellar transport 88 (IFT88) is crucial for craniofacial development in mice and is a candidate gene for human cleft lip and palate.

PubMed

Tian, Hua; Feng, Jifan; Li, Jingyuan; Ho, Thach-Vu; Yuan, Yuan; Liu, Yang; Brindopke, Frederick; Figueiredo, Jane C; Magee, William; Sanchez-Lara, Pedro A; Chai, Yang

2017-03-01

Ciliopathies are pleiotropic human diseases resulting from defects of the primary cilium, and these patients often have cleft lip and palate. IFT88 is required for the assembly and function of the primary cilia, which mediate the activity of key developmental signaling pathways. Through whole exome sequencing of a family of three affected siblings with isolated cleft lip and palate, we discovered that they share a novel missense mutation in IFT88 (c.915G > C, p.E305D), suggesting this gene should be considered a candidate for isolated orofacial clefting. In order to evaluate the function of IFT88 in regulating craniofacial development, we generated Wnt1-Cre;Ift88fl/fl mice to eliminate Ift88 specifically in cranial neural crest (CNC) cells. Wnt1-Cre;Ift88fl/flpups died at birth due to severe craniofacial defects including bilateral cleft lip and palate and tongue agenesis, following the loss of the primary cilia in the CNC-derived palatal mesenchyme. Loss of Ift88 also resulted in a decrease in neural crest cell proliferation during early stages of palatogenesis as well as a downregulation of the Shh signaling pathway in the palatal mesenchyme. Importantly, Osr2KI-Cre;Ift88fl/flmice, in which Ift88 is lost specifically in the palatal mesenchyme, exhibit isolated cleft palate. Taken together, our results demonstrate that IFT88 has a highly conserved function within the primary cilia of the CNC-derived mesenchyme in the lip and palate region in mice and is a strong candidate as an orofacial clefting gene in humans. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A novel form of ciliopathy underlies hyperphagia and obesity in Ankrd26 knockout mice.

PubMed

Acs, Peter; Bauer, Peter O; Mayer, Balazs; Bera, Tapan; Macallister, Rhonda; Mezey, Eva; Pastan, Ira

2015-01-01

Human ciliopathies are genetic disorders caused by mutations in genes responsible for the formation and function of primary cilia. Some are associated with hyperphagia and obesity (e.g., Bardet-Biedl Syndrome, Alström Syndrome), but the mechanisms underlying these problems are not fully understood. The human gene ANKRD26 is located on 10p12, a locus that is associated with some forms of hereditary obesity. Previously, we reported that disruption of this gene causes hyperphagia, obesity and gigantism in mice. In the present study, we looked for the mechanisms that induce hyperphagia in the Ankrd26-/- mice and found defects in primary cilia in regions of the central nervous system that control appetite and energy homeostasis.

Inactivation of Chibby affects function of motile airway cilia

PubMed Central

Voronina, Vera A.; Treuting, Piper; Love, Damon; Grubb, Barbara R.; Hajjar, Adeline M.; Adams, Allison; Li, Feng-Qian; Moon, Randall T.

2009-01-01

Chibby (Cby) is a conserved component of the Wnt–β-catenin pathway. Cby physically interacts with β-catenin to repress its activation of transcription. To elucidate the function of Cby in vertebrates, we generated Cby−/− mice and found that after 2–3 d of weight loss, the majority of mice die before or around weaning. All Cby−/− mice develop rhinitis and sinusitis. When challenged with Pseudomonas aeruginosa isolates, Cby−/− mice are unable to clear the bacteria from the nasal cavity. Notably, Cby−/− mice exhibit a complete absence of mucociliary transport caused by a marked paucity of motile cilia in the nasal epithelium. Moreover, ultrastructural experiments reveal impaired basal body docking to the apical surface of multiciliated cells. In support of these phenotypes, endogenous Cby protein is localized at the base of cilia. As the phenotypes of Cby−/− mice bear striking similarities to primary ciliary dyskinesia, Cby−/− mice may prove to be a useful model for this condition. PMID:19364920

Be together, not the same: Spatiotemporal organization of different cilia types generates distinct transport functions

NASA Astrophysics Data System (ADS)

Nawroth, Janna; Guo, Hanliang; Ruby, Edward; Dabiri, John; McFall-Ngai, Margaret; Kanso, Eva

2016-11-01

Motile cilia are microscopic, hair-like structures on the cell surface that can sense and propel the extracellular fluid environment. Cilia are often thought to be limited to stereotypic morphologies, beat kinematics and non-discriminatory clearance functions, but we find that the spatiotemporal organization of different cilia types and beat behaviors can generate complex flow patterns and transport functions. Here, we present a case study in the Hawaiian bobtail squid where collective ciliary activity and resulting flow fields help recruit symbiont bacteria to the animal host. In particular, we demonstrate empirically and computationally how the squid's internal cilia act like a microfluidic device that actively filters the water for potential bacterial candidates and also provides a sheltered zone allowing for accumulation of mucus and bacteria into a biofilm. Moreover, in this sheltered zone, different cilia-driven flows enhance diffusion of biochemical signals, which could accelerate specific bacteria-host recognition. These results suggest that studying cilia activity on the population level might reveal a diverse range of biological transport and sensing functions. Moreover, understanding cilia as functional building blocks could inspire the design of ciliated robots and devices.

Lack of cilia and differentiation defects in the liver of human foetuses with the Meckel syndrome.

PubMed

Clotman, Frédéric; Libbrecht, Louis; Killingsworth, Murray C; Loo, Christine C K; Roskams, Tania; Lemaigre, Frédéric P

2008-03-01

Meckel syndrome is an autosomal-recessive disease characterized by a combination of renal cysts, anomalies of the central nervous system, polydactyly and ductal plate malformations (DPM), which are hepatic anomalies consisting of excessive and abnormal foetal biliary structures. Among the genomic loci associated with Meckel syndrome, mutations in four genes were recently identified. These genes code for proteins associated with primary cilia and are possibly involved in cell differentiation. The aim of the present work was to investigate the formation of the primary cilia and the differentiation of the hepatic cells in foetuses with Meckel syndrome. Sections of livers from human foetuses with Meckel syndrome were analysed by immunofluorescence, immunohistochemistry and electron microscopy. The primary cilia of the biliary cells were absent in some Meckel foetuses, but were present in others. In addition, defects in hepatic differentiation were observed in Meckel livers, as evidenced by the presence of hybrid cells co-expressing hepatocytic and biliary markers. Defects in cilia formation occur in some Meckel livers, and most cases show DPM associated with abnormal hepatic cell differentiation. Because differentiation precedes the formation of the cilia during liver development, we propose that defective differentiation may constitute the initial defect in the liver of Meckel syndrome foetuses.

Functional characterization of putative cilia genes by high-content analysis

PubMed Central

Lai, Cary K.; Gupta, Nidhi; Wen, Xiaohui; Rangell, Linda; Chih, Ben; Peterson, Andrew S.; Bazan, J. Fernando; Li, Li; Scales, Suzie J.

2011-01-01

Cilia are microtubule-based protrusions from the cell surface that are involved in a number of essential signaling pathways, yet little is known about many of the proteins that regulate their structure and function. A number of putative cilia genes have been identified by proteomics and comparative sequence analyses, but functional data are lacking for the vast majority. We therefore monitored the effects in three cell lines of small interfering RNA (siRNA) knockdown of 40 of these genes by high-content analysis. We assayed cilia number, length, and transport of two different cargoes (membranous serotonin receptor 6-green fluorescent protein [HTR6-GFP] and the endogenous Hedgehog [Hh] pathway transcription factor Gli3) by immunofluorescence microscopy; and cilia function using a Gli-luciferase Hh signaling assay. Hh signaling was most sensitive to perturbations, with or without visible structural cilia defects. Validated hits include Ssa2 and mC21orf2 with ciliation defects; Ift46 with short cilia; Ptpdc1 and Iqub with elongated cilia; and Arl3, Nme7, and Ssna1 with distinct ciliary transport but not length defects. Our data confirm various ciliary roles for several ciliome proteins and show it is possible to uncouple ciliary cargo transport from cilia formation in vertebrates. PMID:21289087

The Orphan G Protein-coupled Receptor Gpr175 (Tpra40) Enhances Hedgehog Signaling by Modulating cAMP Levels.

PubMed

Singh, Jaskirat; Wen, Xiaohui; Scales, Suzie J

2015-12-04

The Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as Tpra1 or Tpra40: transmembrane protein, adipocyte associated 1 or of 40 kDa), which also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling. Interaction experiments place Gpr175 at the level of PKA and upstream of the Gαi component of heterotrimeric G proteins, which itself localizes to cilia and can modulate Hh signaling. Gpr175 or Gαi1 depletion leads to increases in cellular cAMP levels and in Gli3 processing into its repressor form. Thus we propose that Gpr175 coupled to Gαi1 normally functions to inhibit the production of cAMP by adenylyl cyclase upon Hh stimulation, thus maximizing signaling by turning off PKA activity and hence Gli3 repressor formation. Taken together our data suggest that Gpr175 is a novel positive regulator of the Hh signaling pathway. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Oscillatory fluid flow influences primary cilia and microtubule mechanics.

PubMed

Espinha, Lina C; Hoey, David A; Fernandes, Paulo R; Rodrigues, Hélder C; Jacobs, Christopher R

2014-07-01

Many tissues are sensitive to mechanical stimuli; however, the mechanotransduction mechanism used by cells remains unknown in many cases. The primary cilium is a solitary, immotile microtubule-based extension present on nearly every mammalian cell which extends from the basal body. The cilium is a mechanosensitive organelle and has been shown to transduce fluid flow-induced shear stress in tissues, such as the kidney and bone. The majority of microtubules assemble from the mother centriole (basal body), contributing significantly to the anchoring of the primary cilium. Several studies have attempted to quantify the number of microtubules emanating from the basal body and the results vary depending on the cell type. It has also been shown that cellular response to shear stress depends on microtubular integrity. This study hypothesizes that changing the microtubule attachment of primary cilia in response to a mechanical stimulus could change primary cilia mechanics and, possibly, mechanosensitivity. Oscillatory fluid flow was applied to two different cell types and the microtubule attachment to the ciliary base was quantified. For the first time, an increase in microtubules around primary cilia both with time and shear rate in response to oscillatory fluid flow stimulation was demonstrated. Moreover, it is presented that the primary cilium is required for this loading-induced cellular response. This study has demonstrated a new role for the cilium in regulating alterations in the cytoplasmic microtubule network in response to mechanical stimulation, and therefore provides a new insight into how cilia may regulate its mechanics and thus the cells mechanosensitivity. Copyright © 2014 Wiley Periodicals, Inc.

A novel form of ciliopathy underlies hyperphagia and obesity in Ankrd26 knockout mice

PubMed Central

Acs, Peter; Bauer, Peter O.; Mayer, Balazs; Bera, Tapan; Macallister, Rhonda; Pastan, Ira

2015-01-01

Human ciliopathies are genetic disorders caused by mutations in genes responsible for the formation and function of primary cilia. Some are associated with hyperphagia and obesity (e.g., Bardet–Biedl Syndrome, Alström Syndrome), but the mechanisms underlying these problems are not fully understood. The human gene ANKRD26 is located on 10p12, a locus that is associated with some forms of hereditary obesity. Previously, we reported that disruption of this gene causes hyperphagia, obesity and gigantism in mice. In the present study, we looked for the mechanisms that induce hyperphagia in the Ankrd26−/− mice and found defects in primary cilia in regions of the central nervous system that control appetite and energy homeostasis. PMID:24633808

Methods for Studying Ciliary-Mediated Chemoresponse in Paramecium.

PubMed

Valentine, Megan Smith; Van Houten, Judith L

2016-01-01

Paramecium is a useful model organism for the study of ciliary-mediated chemical sensing and response. Here we describe ways to take advantage of Paramecium to study chemoresponse.Unicellular organisms like the ciliated protozoan Paramecium sense and respond to chemicals in their environment (Van Houten, Ann Rev Physiol 54:639-663, 1992; Van Houten, Trends Neurosci 17:62-71, 1994). A thousand or more cilia that cover Paramecium cells serve as antennae for chemical signals, similar to ciliary function in a large variety of metazoan cell types that have primary or motile cilia (Berbari et al., Curr Biol 19(13):R526-R535, 2009; Singla V, Reiter J, Science 313:629-633, 2006). The Paramecium cilia also produce the motor output of the detection of chemical cues by controlling swimming behavior. Therefore, in Paramecium the cilia serve multiple roles of detection and response.We present this chapter in three sections to describe the methods for (1) assaying populations of cells for their behavioral responses to chemicals (attraction and repulsion), (2) characterization of the chemoreceptors and associated channels of the cilia using proteomics and binding assays, and (3) electrophysiological analysis of individual cells' responses to chemicals. These methods are applied to wild type cells, mutants, transformed cells that express tagged proteins, and cells depleted of gene products by RNA Interference (RNAi).

Computer-assisted image analysis of human cilia and Chlamydomonas flagella reveals both similarities and differences in axoneme structure.

PubMed

O'Toole, Eileen T; Giddings, Thomas H; Porter, Mary E; Ostrowski, Lawrence E

2012-08-01

In the past decade, investigations from several different fields have revealed the critical role of cilia in human health and disease. Because of the highly conserved nature of the basic axonemal structure, many different model systems have proven useful for the study of ciliopathies, especially the unicellular, biflagellate green alga Chlamydomonas reinhardtii. Although the basic axonemal structure of cilia and flagella is highly conserved, these organelles often perform specialized functions unique to the cell or tissue in which they are found. These differences in function are likely reflected in differences in structural organization. In this work, we directly compare the structure of isolated axonemes from human cilia and Chlamydomonas flagella to identify similarities and differences that potentially play key roles in determining their functionality. Using transmission electron microscopy and 2D image averaging techniques, our analysis has confirmed the overall structural similarity between these two species, but also revealed clear differences in the structure of the outer dynein arms, the central pair projections, and the radial spokes. We also show how the application of 2D image averaging can clarify the underlying structural defects associated with primary ciliary dyskinesia (PCD). Overall, our results document the remarkable similarity between these two structures separated evolutionarily by over a billion years, while highlighting several significant differences, and demonstrate the potential of 2D image averaging to improve the diagnosis and understanding of PCD. Copyright © 2012 Wiley Periodicals, Inc.

ciliaFA: a research tool for automated, high-throughput measurement of ciliary beat frequency using freely available software

PubMed Central

2012-01-01

Background Analysis of ciliary function for assessment of patients suspected of primary ciliary dyskinesia (PCD) and for research studies of respiratory and ependymal cilia requires assessment of both ciliary beat pattern and beat frequency. While direct measurement of beat frequency from high-speed video recordings is the most accurate and reproducible technique it is extremely time consuming. The aim of this study was to develop a freely available automated method of ciliary beat frequency analysis from digital video (AVI) files that runs on open-source software (ImageJ) coupled to Microsoft Excel, and to validate this by comparison to the direct measuring high-speed video recordings of respiratory and ependymal cilia. These models allowed comparison to cilia beating between 3 and 52 Hz. Methods Digital video files of motile ciliated ependymal (frequency range 34 to 52 Hz) and respiratory epithelial cells (frequency 3 to 18 Hz) were captured using a high-speed digital video recorder. To cover the range above between 18 and 37 Hz the frequency of ependymal cilia were slowed by the addition of the pneumococcal toxin pneumolysin. Measurements made directly by timing a given number of individual ciliary beat cycles were compared with those obtained using the automated ciliaFA system. Results The overall mean difference (± SD) between the ciliaFA and direct measurement high-speed digital imaging methods was −0.05 ± 1.25 Hz, the correlation coefficient was shown to be 0.991 and the Bland-Altman limits of agreement were from −1.99 to 1.49 Hz for respiratory and from −2.55 to 3.25 Hz for ependymal cilia. Conclusions A plugin for ImageJ was developed that extracts pixel intensities and performs fast Fourier transformation (FFT) using Microsoft Excel. The ciliaFA software allowed automated, high throughput measurement of respiratory and ependymal ciliary beat frequency (range 3 to 52 Hz) and avoids operator error due to selection bias. We have included free access to the ciliaFA plugin and installation instructions in Additional file 1 accompanying this manuscript that other researchers may use. PMID:23351276

Recessive HYDIN Mutations Cause Primary Ciliary Dyskinesia without Randomization of Left-Right Body Asymmetry

PubMed Central

Olbrich, Heike; Schmidts, Miriam; Werner, Claudius; Onoufriadis, Alexandros; Loges, Niki T.; Raidt, Johanna; Banki, Nora Fanni; Shoemark, Amelia; Burgoyne, Tom; Al Turki, Saeed; Hurles, Matthew E.; Köhler, Gabriele; Schroeder, Josef; Nürnberg, Gudrun; Nürnberg, Peter; Chung, Eddie M.K.; Reinhardt, Richard; Marthin, June K.; Nielsen, Kim G.; Mitchison, Hannah M.; Omran, Heymut

2012-01-01

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder characterized by defective cilia and flagella motility. Chronic destructive-airway disease is caused by abnormal respiratory-tract mucociliary clearance. Abnormal propulsion of sperm flagella contributes to male infertility. Genetic defects in most individuals affected by PCD cause randomization of left-right body asymmetry; approximately half show situs inversus or situs ambiguous. Almost 70 years after the hy3 mouse possessing Hydin mutations was described as a recessive hydrocephalus model, we report HYDIN mutations in PCD-affected persons without hydrocephalus. By homozygosity mapping, we identified a PCD-associated locus, chromosomal region 16q21-q23, which contains HYDIN. However, a nearly identical 360 kb paralogous segment (HYDIN2) in chromosomal region 1q21.1 complicated mutational analysis. In three affected German siblings linked to HYDIN, we identified homozygous c.3985G>T mutations that affect an evolutionary conserved splice acceptor site and that subsequently cause aberrantly spliced transcripts predicting premature protein termination in respiratory cells. Parallel whole-exome sequencing identified a homozygous nonsense HYDIN mutation, c.922A>T (p.Lys307∗), in six individuals from three Faroe Island PCD-affected families that all carried an 8.8 Mb shared haplotype across HYDIN, indicating an ancestral founder mutation in this isolated population. We demonstrate by electron microscopy tomography that, consistent with the effects of loss-of-function mutations, HYDIN mutant respiratory cilia lack the C2b projection of the central pair (CP) apparatus; similar findings were reported in Hydin-deficient Chlamydomonas and mice. High-speed videomicroscopy demonstrated markedly reduced beating amplitudes of respiratory cilia and stiff sperm flagella. Like the hy3 mouse model, all nine PCD-affected persons had normal body composition because nodal cilia function is apparently not dependent on the function of the CP apparatus. PMID:23022101

New Roles of the Primary Cilium in Autophagy

PubMed Central

Ávalos, Yenniffer; Peña-Oyarzun, Daniel; Budini, Mauricio

2017-01-01

The primary cilium is a nonmotile organelle that emanates from the surface of multiple cell types and receives signals from the environment to regulate intracellular signaling pathways. The presence of cilia, as well as their length, is important for proper cell function; shortened, elongated, or absent cilia are associated with pathological conditions. Interestingly, it has recently been shown that the molecular machinery involved in autophagy, the process of recycling of intracellular material to maintain cellular and tissue homeostasis, participates in ciliogenesis. Cilium-dependent signaling is necessary for autophagosome formation and, conversely, autophagy regulates both ciliogenesis and cilium length by degrading specific ciliary proteins. Here, we will discuss the relationship that exists between the two processes at the cellular and molecular level, highlighting what is known about the effects of ciliary dysfunction in the control of energy homeostasis in some ciliopathies. PMID:28913352

New Roles of the Primary Cilium in Autophagy.

PubMed

Ávalos, Yenniffer; Peña-Oyarzun, Daniel; Budini, Mauricio; Morselli, Eugenia; Criollo, Alfredo

2017-01-01

The primary cilium is a nonmotile organelle that emanates from the surface of multiple cell types and receives signals from the environment to regulate intracellular signaling pathways. The presence of cilia, as well as their length, is important for proper cell function; shortened, elongated, or absent cilia are associated with pathological conditions. Interestingly, it has recently been shown that the molecular machinery involved in autophagy, the process of recycling of intracellular material to maintain cellular and tissue homeostasis, participates in ciliogenesis. Cilium-dependent signaling is necessary for autophagosome formation and, conversely, autophagy regulates both ciliogenesis and cilium length by degrading specific ciliary proteins. Here, we will discuss the relationship that exists between the two processes at the cellular and molecular level, highlighting what is known about the effects of ciliary dysfunction in the control of energy homeostasis in some ciliopathies.

Sperm Associated Antigen 6 (SPAG6) Regulates Fibroblast Cell Growth, Morphology, Migration and Ciliogenesis

PubMed Central

Li, Wei; Mukherjee, Abir; Wu, Jinhua; Zhang, Ling; Teves, Maria E.; Li, Hongfei; Nambiar, Shanti; Henderson, Scott C.; Horwitz, Alan R.; Strauss III, Jerome F.; Fang, Xianjun; Zhang, Zhibing

2015-01-01

Mammalian Spag6 is the orthologue of Chlamydomonas PF16, which encodes a protein localized in the axoneme central apparatus, and regulates flagella/cilia motility. Most Spag6-deficient mice are smaller in size than their littermates. Because SPAG6 decorates microtubules, we hypothesized that SPAG6 has other roles related to microtubule function besides regulating flagellar/cilia motility. Mouse embryonic fibroblasts (MEFs) were isolated from Spag6-deficient and wild-type embryos for these studies. Both primary and immortalized Spag6-deficient MEFs proliferated at a much slower rate than the wild-type MEFs, and they had a larger surface area. Re-expression of SPAG6 in the Spag6-deficient MEFs rescued the abnormal cell morphology. Spag6-deficient MEFs were less motile than wild-type MEFs, as shown by both chemotactic analysis and wound-healing assays. Spag6-deficient MEFs also showed reduced adhesion associated with a non-polarized F-actin distribution. Multiple centrosomes were observed in the Spag6-deficient MEF cultures. The percentage of cells with primary cilia was significantly reduced compared to the wild-type MEFs, and some Spag6-deficient MEFs developed multiple cilia. Furthermore, SPAG6 selectively increased expression of acetylated tubulin, a microtubule stability marker. The Spag6-deficient MEFs were more sensitive to paclitaxel, a microtubule stabilizer. Our studies reveal new roles for SPAG6 in modulation of cell morphology, proliferation, migration, and ciliogenesis. PMID:26585507

SEPT9_v1 Functions in Breast Cancer Cell Division

DTIC Science & Technology

2012-01-01

the regulation and function of septin filaments, and define new mechanisms regulating important cellular functions. BODY: 1). Study the effects...ciliogenesis. However, mechanisms for retaining these proteins and lipids in the primary cilia are not clear. We directly tested the presence of a diffusion...polarity and morphogenesis;  Defined mechanisms involving the roles of septins and microtubules in vesicle trafficking and epithelial morphogenesis

Clinical and genetic aspects of primary ciliary dyskinesia/Kartagener syndrome.

PubMed

Leigh, Margaret W; Pittman, Jessica E; Carson, Johnny L; Ferkol, Thomas W; Dell, Sharon D; Davis, Stephanie D; Knowles, Michael R; Zariwala, Maimoona A

2009-07-01

Primary ciliary dyskinesia is a genetically heterogeneous disorder of motile cilia. Most of the disease-causing mutations identified to date involve the heavy (dynein axonemal heavy chain 5) or intermediate(dynein axonemal intermediate chain 1) chain dynein genes in ciliary outer dynein arms, although a few mutations have been noted in other genes. Clinical molecular genetic testing for primary ciliary dyskinesia is available for the most common mutations. The respiratory manifestations of primary ciliary dyskinesia (chronic bronchitis leading to bronchiectasis, chronic rhino-sinusitis, and chronic otitis media)reflect impaired mucociliary clearance owing to defective axonemal structure. Ciliary ultrastructural analysis in most patients (>80%) reveals defective dynein arms, although defects in other axonemal components have also been observed. Approximately 50% of patients with primary ciliary dyskinesia have laterality defects (including situs inversus totalis and, less commonly, heterotaxy, and congenital heart disease),reflecting dysfunction of embryological nodal cilia. Male infertility is common and reflects defects in sperm tail axonemes. Most patients with primary ciliary dyskinesia have a history of neonatal respiratory distress, suggesting that motile cilia play a role in fluid clearance during the transition from a fetal to neonatal lung. Ciliopathies involving sensory cilia, including autosomal dominant or recessive polycystic kidney disease, Bardet-Biedl syndrome, and Alstrom syndrome, may have chronic respiratory symptoms and even bronchiectasis suggesting clinical overlap with primary ciliary dyskinesia.

Force-response considerations in ciliary mechanosensation.

PubMed

Resnick, Andrew; Hopfer, Ulrich

2007-08-15

Considerable experimental evidence indicates that the primary, nonmotile cilium is a mechanosensory organelle in several epithelial cell types. As the relationship between cellular responses and nature and magnitude of applied forces is not well understood, we have investigated the effects of exposure of monolayers of renal collecting duct chief cells to orbital shaking and quantified the forces incident on cilia. An exposure of 24 h of these cells to orbital shaking resulted in a decrease of amiloride-sensitive sodium current by approximately 60% and ciliary length by approximately 30%. The sensitivity of the sodium current to shaking was dependent on intact cilia. The drag force on cilia due to induced fluid flow during orbital shaking was estimated at maximally 5.2x10(-3) pN at 2 Hz, approximately 4 times that of thermal noise. The major structural feature of cilia contributing to their sensitivity appears to be ciliary length. As more than half of the total drag force is exerted on the ciliary cap, one function of the slender stalk may be to expose the cap to greater drag force. Regardless, the findings indicate that the cilium is a mechanosensory organelle with a sensitivity much lower than previously recognized.

ARL2BP, a protein linked to Retinitis Pigmentosa, is needed for normal photoreceptor cilia doublets and outer segment structure.

PubMed

Moye, Abigail R; Singh, Ratnesh; Kimler, Victoria A; Dilan, Tanya L; Munezero, Daniella; Saravanan, Thamaraiselvi; Goldberg, Andrew F X; Ramamurthy, Visvanathan

2018-05-02

The outer segment (OS) of photoreceptor cells is an elaboration of a primary cilium with organized stacks of membranous discs that contain the proteins needed for phototransduction and vision. Though cilia formation and function has been well characterized, little is known about the role of cilia in the development of photoreceptor OS. Nevertheless, progress has been made by studying mutations in ciliary proteins which often result in malformed outer segments and lead to blinding diseases. To investigate how ciliary proteins contribute to outer segment formation, we generated a knockout mouse model for ARL2BP, a ciliary protein linked to Retinitis Pigmentosa. The knockout mice display an early and progressive reduction in visual response. Prior to photoreceptor degeneration we observed disorganization of the photoreceptor OS, with vertically aligned discs and shortened axonemes. Interestingly, ciliary doublet microtubule structure was also impaired, displaying open B-tubule doublets, paired with loss of singlet microtubules. Based on results from this study, we conclude that ARL2BP is necessary for photoreceptor cilia doublet formation and axoneme elongation, which is required for outer segment morphogenesis and vision.

Primary Cilia in Breast Cancer Progression

DTIC Science & Technology

2010-06-01

differential distribution of acetylated and detyrosinated alpha-tubulin in the microtubular cytoskeleton and primary cilia of hyaline cartilage ...disrupted. Nevertheless, minimal disruption to normal mammary development was observed. Studies to determine the role of PC in tumor progression are...the role of PC in normal mammary development or tumor formation. The purpose of this synergistic study was to begin to address the role of this

A Wnt/beta-catenin pathway antagonist Chibby binds Cenexin at the distal end of mother centrioles and functions in primary cilia formation.

PubMed

Steere, Nathan; Chae, Vanessa; Burke, Michael; Li, Feng-Qian; Takemaru, Ken-ichi; Kuriyama, Ryoko

2012-01-01

The mother centriole of the centrosome is distinguished from immature daughter centrioles by the presence of accessory structures (distal and subdistal appendages), which play an important role in the organization of the primary cilium in quiescent cells. Primary cilia serve as sensory organelles, thus have been implicated in mediating intracellular signal transduction pathways. Here we report that Chibby (Cby), a highly conserved antagonist of the Wnt/β-catenin pathway, is a centriolar component specifically located at the distal end of the mother centriole and essential for assembly of the primary cilium. Cby appeared as a discrete dot in the middle of a ring-like structure revealed by staining with a distal appendage component of Cep164. Cby interacted with one of the appendage components, Cenexin (Cnx), which thereby abrogated the inhibitory effect of Cby on β-catenin-mediated transcriptional activation in a dose-dependent manner. Cby and Cnx did not precisely align, as Cby was detected at a more distal position than Cnx. Cnx emerged earlier than Cby during the cell cycle and was required for recruitment of Cby to the mother centriole. However, Cby was dispensable for Cnx localization to the centriole. During massive centriogenesis in in vitro cultured mouse tracheal epithelial cells, Cby and Cnx were expressed in a similar pattern, which was coincident with the expression of Foxj1. Our results suggest that Cby plays an important role in organization of both primary and motile cilia in collaboration with Cnx.

Low-intensity pulsed ultrasound stimulation promotes osteoblast differentiation through hedgehog signaling.

PubMed

Matsumoto, Kenichi; Shimo, Tsuyoshi; Kurio, Naito; Okui, Tatsuo; Ibaragi, Soichiro; Kunisada, Yuki; Obata, Kyoichi; Masui, Masanori; Pai, Pang; Horikiri, Yuu; Yamanaka, Nobuyuki; Takigawa, Masaharu; Sasaki, Akira

2018-06-01

Low-intensity pulsed ultrasound (LIPUS) has been used as an adjunct to fracture healing therapies, but the mechanisms underlying its action are not known. We reported that sonic hedgehog (SHH) signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture. Mechanical stimulation is a crucial factor in bone remodeling, and it is related to the primary cilia as a sensor of hedgehog signaling. Here we observed that LIPUS promoted callus formation in accord with Gli2-positive cells after 14 days at the mouse femur fractured site compared with a control group. An immunofluorescence analysis showed that the numbers of primary cilia and cilia/osterix double-positive osteoblasts were increased at the fracture site by LIPUS. LIPUS stimulated not only the number and the length of primary cilia, but also the levels of ciliated protein, Ift88 mRNA, and SHH, Gli1, and Gli2 in MC3T3-E1 cells. Further experiments revealed that LIPUS stimulated osteogenic differentiation in the presence of smoothened agonist (SAG) treatment. These results indicate that LIPUS stimulates osteogenic differentiation and the maturation of osteoblasts by a primary cilium-mediated activation of hedgehog signaling. © 2017 Wiley Periodicals, Inc.

Neuropeptide Y family receptors traffic via the Bardet-Biedl syndrome pathway to signal in neuronal primary cilia.

PubMed

Loktev, Alexander V; Jackson, Peter K

2013-12-12

Human monogenic obesity syndromes, including Bardet-Biedl syndrome (BBS), implicate neuronal primary cilia in regulation of energy homeostasis. Cilia in hypothalamic neurons have been hypothesized to sense and regulate systemic energy status, but the molecular mechanism of this signaling remains unknown. Here, we report a comprehensive localization screen of 42 G-protein-coupled receptors (GPCR) revealing seven ciliary GPCRs, including the neuropeptide Y (NPY) receptors NPY2R and NPY5R. We show that mice modeling BBS disease or obese tubby mice fail to localize NPY2R to cilia in the hypothalamus and that BBS mutant mice fail to activate c-fos or decrease food intake in response to the NPY2R ligand PYY3-36. We find that cells with ciliary NPY2R show augmented PYY3-36-dependent cAMP signaling. Our data demonstrate that ciliary targeting of NPY receptors is important for controlling energy balance in mammals, revealing a physiologically defined ligand-receptor pathway signaling within neuronal cilia. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier

PubMed Central

Breslow, David K.; Koslover, Elena F.; Seydel, Federica; Spakowitz, Andrew J.

2013-01-01

Specific proteins are concentrated within primary cilia, whereas others remain excluded. To understand the mechanistic basis of entry into cilia, we developed an in vitro assay using cells in which the plasma membrane was permeabilized, but the ciliary membrane was left intact. Using a diffusion-to-capture system and quantitative analysis, we find that proteins >9 nm in diameter (∼100 kD) are restricted from entering cilia, and we confirm these findings in vivo. Interference with the nuclear pore complex (NPC) or the actin cytoskeleton in permeabilized cells demonstrated that the ciliary diffusion barrier is mechanistically distinct from those of the NPC or the axon initial segment. Moreover, applying a mass transport model to this system revealed diffusion coefficients for soluble and membrane proteins within cilia that are compatible with rapid exploration of the ciliary space in the absence of active transport. Our results indicate that large proteins require active transport for entry into cilia but not necessarily for movement inside cilia. PMID:24100294

Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells

PubMed Central

Li, Li; Grausam, Katie B.; Wang, Jun; Lun, Melody P.; Ohli, Jasmin; Lidov, Hart G. W.; Calicchio, Monica L.; Zeng, Erliang; Salisbury, Jeffrey L.; Wechsler-Reya, Robert J.; Lehtinen, Maria K.; Schüller, Ulrich; Zhao, Haotian

2016-01-01

Aberrant Notch signaling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly pediatric brain neoplasms. We developed animal models of CP tumours by inducing sustained expression of Notch1 that recapitulate properties of human CP tumours with aberrant NOTCH signaling. Whole transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate diffferentiation. A Shh-driven signaling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from mono-ciliated progenitors in the roof plate characterized by elevated Notch signaling. Abnormal SHH signaling and distinct ciliogenesis are detected in human CP tumours, suggesting SHH pathway and cilia differentiation as potential therapeutic avenues. PMID:26999738

Mutations in CSPP1 lead to classical Joubert syndrome.

PubMed

Akizu, Naiara; Silhavy, Jennifer L; Rosti, Rasim Ozgur; Scott, Eric; Fenstermaker, Ali G; Schroth, Jana; Zaki, Maha S; Sanchez, Henry; Gupta, Neerja; Kabra, Madhulika; Kara, Majdi; Ben-Omran, Tawfeg; Rosti, Basak; Guemez-Gamboa, Alicia; Spencer, Emily; Pan, Roger; Cai, Na; Abdellateef, Mostafa; Gabriel, Stacey; Halbritter, Jan; Hildebrandt, Friedhelm; van Bokhoven, Hans; Gunel, Murat; Gleeson, Joseph G

2014-01-02

Joubert syndrome and related disorders (JSRDs) are genetically heterogeneous and characterized by a distinctive mid-hindbrain malformation. Causative mutations lead to primary cilia dysfunction, which often results in variable involvement of other organs such as the liver, retina, and kidney. We identified predicted null mutations in CSPP1 in six individuals affected by classical JSRDs. CSPP1 encodes a protein localized to centrosomes and spindle poles, as well as to the primary cilium. Despite the known interaction between CSPP1 and nephronophthisis-associated proteins, none of the affected individuals in our cohort presented with kidney disease, and further, screening of a large cohort of individuals with nephronophthisis demonstrated no mutations. CSPP1 is broadly expressed in neural tissue, and its encoded protein localizes to the primary cilium in an in vitro model of human neurogenesis. Here, we show abrogated protein levels and ciliogenesis in affected fibroblasts. Our data thus suggest that CSPP1 is involved in neural-specific functions of primary cilia. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

The Roles of Primary Cilia in Cardiovascular System

DTIC Science & Technology

2015-10-01

defect, oral facial syndrome, obesity , hypertension and others [60]. Primary cilia can be activated by bending through perfusing cells with fluid...synthase, was found to be elevated in PKD patients [125]. The correlation between hypertension and kidney volume occurs in the early childhood stages...involvement of centrosome and basal body dysfunction in the pathogenesis of obesity , insulin resistance, and type 2 diabetes. Diabetes 2005; 54(5

Evolution: Tracing the origins of centrioles, cilia, and flagella.

PubMed

Carvalho-Santos, Zita; Azimzadeh, Juliette; Pereira-Leal, José B; Bettencourt-Dias, Mónica

2011-07-25

Centrioles/basal bodies (CBBs) are microtubule-based cylindrical organelles that nucleate the formation of centrosomes, cilia, and flagella. CBBs, cilia, and flagella are ancestral structures; they are present in all major eukaryotic groups. Despite the conservation of their core structure, there is variability in their architecture, function, and biogenesis. Recent genomic and functional studies have provided insight into the evolution of the structure and function of these organelles.

Update on oral-facial-digital syndromes (OFDS).

PubMed

Franco, Brunella; Thauvin-Robinet, Christel

2016-01-01

Oral-facial-digital syndromes (OFDS) represent a heterogeneous group of rare developmental disorders affecting the mouth, the face and the digits. Additional signs may involve brain, kidneys and other organs thus better defining the different clinical subtypes. With the exception of OFD types I and VIII, which are X-linked, the majority of OFDS is transmitted as an autosomal recessive syndrome. A number of genes have already found to be mutated in OFDS and most of the encoded proteins are predicted or proven to be involved in primary cilia/basal body function. Preliminary data indicate a physical interaction among some of those proteins and future studies will clarify whether all OFDS proteins are part of a network functionally connected to cilia. Mutations in some of the genes can also lead to other types of ciliopathies with partially overlapping phenotypes, such as Joubert syndrome (JS) and Meckel syndrome (MKS), supporting the concept that cilia-related diseases might be a continuous spectrum of the same phenotype with different degrees of severity. To date, seven of the described OFDS still await a molecular definition and two unclassified forms need further clinical and molecular validation. Next-generation sequencing (NGS) approaches are expected to shed light on how many OFDS geneticists should consider while evaluating oral-facial-digital cases. Functional studies will establish whether the non-ciliary functions of the transcripts mutated in OFDS might contribute to any of the phenotypic abnormalities observed in OFDS.

Structural and Functional Recovery of Sensory Cilia in C. elegans IFT Mutants upon Aging.

PubMed

Cornils, Astrid; Maurya, Ashish K; Tereshko, Lauren; Kennedy, Julie; Brear, Andrea G; Prahlad, Veena; Blacque, Oliver E; Sengupta, Piali

2016-12-01

The majority of cilia are formed and maintained by the highly conserved process of intraflagellar transport (IFT). Mutations in IFT genes lead to ciliary structural defects and systemic disorders termed ciliopathies. Here we show that the severely truncated sensory cilia of hypomorphic IFT mutants in C. elegans transiently elongate during a discrete period of adult aging leading to markedly improved sensory behaviors. Age-dependent restoration of cilia morphology occurs in structurally diverse cilia types and requires IFT. We demonstrate that while DAF-16/FOXO is dispensable, the age-dependent suppression of cilia phenotypes in IFT mutants requires cell-autonomous functions of the HSF1 heat shock factor and the Hsp90 chaperone. Our results describe an unexpected role of early aging and protein quality control mechanisms in suppressing ciliary phenotypes of IFT mutants, and suggest possible strategies for targeting subsets of ciliopathies.

Structural and Functional Recovery of Sensory Cilia in C. elegans IFT Mutants upon Aging

PubMed Central

Kennedy, Julie; Brear, Andrea G.; Prahlad, Veena; Blacque, Oliver E.; Sengupta, Piali

2016-01-01

The majority of cilia are formed and maintained by the highly conserved process of intraflagellar transport (IFT). Mutations in IFT genes lead to ciliary structural defects and systemic disorders termed ciliopathies. Here we show that the severely truncated sensory cilia of hypomorphic IFT mutants in C. elegans transiently elongate during a discrete period of adult aging leading to markedly improved sensory behaviors. Age-dependent restoration of cilia morphology occurs in structurally diverse cilia types and requires IFT. We demonstrate that while DAF-16/FOXO is dispensable, the age-dependent suppression of cilia phenotypes in IFT mutants requires cell-autonomous functions of the HSF1 heat shock factor and the Hsp90 chaperone. Our results describe an unexpected role of early aging and protein quality control mechanisms in suppressing ciliary phenotypes of IFT mutants, and suggest possible strategies for targeting subsets of ciliopathies. PMID:27906968

Statistical Modeling of an Optically Trapped Cilium

NASA Astrophysics Data System (ADS)

Flaherty, Justin; Resnick, Andrew

We explore, analytically and experimentally, the stochastic dynamics of a biologically significant slender microcantilever, the primary cilium, held within an optical trap. Primary cilia are cellular organelles, present on most vertebrate cells, hypothesized to function as a fluid flow sensor. The mechanical properties of a cilium remain incompletely characterized. Optical trapping is an ideal method to probe the mechanical response of a cilium due to the spatial localization and non-contact nature of the applied force. However, analysis of an optically trapped cilium is complicated both by the geometry of a cilium and boundary conditions. Here, we present experimentally measured mean-squared displacement data of trapped cilia where the trapping force is oppositely directed to the elastic restoring force of the ciliary axoneme, analytical modeling results deriving the mean-squared displacement of a trapped cilium using the Langevin approach, and apply our analytical results to the experimental data. We demonstrate that mechanical properties of the cilium can be accurately determined and efficiently extracted from the data using our model. It is hoped that improved measurements will result in deeper understanding of the biological function of cellular flow sensing by this organelle.

Novel Insights into the Development and Function of Cilia Using the Advantages of the Paramecium Cell and Its Many Cilia

PubMed Central

Yano, Junji; Valentine, Megan S.; Van Houten, Judith L.

2015-01-01

Paramecium species, especially P. tetraurelia and caudatum, are model organisms for modern research into the form and function of cilia. In this review, we focus on the ciliary ion channels and other transmembrane proteins that control the beat frequency and wave form of the cilium by controlling the signaling within the cilium. We put these discussions in the context of the advantages that Paramecium brings to the understanding of ciliary motility: mutants for genetic dissections of swimming behavior, electrophysiology, structural analysis, abundant cilia for biochemistry and modern proteomics, genomics and molecular biology. We review the connection between behavior and physiology, which allows the cells to broadcast the function of their ciliary channels in real time. We build a case for the important insights and advantages that this model organism continues to bring to the study of cilia. PMID:26230712

Novel Insights into the Development and Function of Cilia Using the Advantages of the Paramecium Cell and Its Many Cilia.

PubMed

Yano, Junji; Valentine, Megan S; Van Houten, Judith L

2015-07-29

Paramecium species, especially P. tetraurelia and caudatum, are model organisms for modern research into the form and function of cilia. In this review, we focus on the ciliary ion channels and other transmembrane proteins that control the beat frequency and wave form of the cilium by controlling the signaling within the cilium. We put these discussions in the context of the advantages that Paramecium brings to the understanding of ciliary motility: mutants for genetic dissections of swimming behavior, electrophysiology, structural analysis, abundant cilia for biochemistry and modern proteomics, genomics and molecular biology. We review the connection between behavior and physiology, which allows the cells to broadcast the function of their ciliary channels in real time. We build a case for the important insights and advantages that this model organism continues to bring to the study of cilia.

Transcriptional program of ciliated epithelial cells reveals new cilium and centrosome components and links to human disease.

PubMed

Hoh, Ramona A; Stowe, Timothy R; Turk, Erin; Stearns, Tim

2012-01-01

Defects in the centrosome and cilium are associated with a set of human diseases having diverse phenotypes. To further characterize the components that define the function of these organelles we determined the transcriptional profile of multiciliated tracheal epithelial cells. Cultures of mouse tracheal epithelial cells undergoing differentiation in vitro were derived from mice expressing GFP from the ciliated-cell specific FOXJ1 promoter (FOXJ1:GFP). The transcriptional profile of ciliating GFP+ cells from these cultures was defined at an early and a late time point during differentiation and was refined by subtraction of the profile of the non-ciliated GFP- cells. We identified 649 genes upregulated early, when most cells were forming basal bodies, and 73 genes genes upregulated late, when most cells were fully ciliated. Most, but not all, of known centrosome proteins are transcriptionally upregulated early, particularly Plk4, a master regulator of centriole formation. We found that three genes associated with human disease states, Mdm1, Mlf1, and Dyx1c1, are upregulated during ciliogenesis and localize to centrioles and cilia. This transcriptome for mammalian multiciliated epithelial cells identifies new candidate centrosome and cilia proteins, highlights similarities between components of motile and primary cilia, and identifies new links between cilia proteins and human disease.

Transcriptional Program of Ciliated Epithelial Cells Reveals New Cilium and Centrosome Components and Links to Human Disease

PubMed Central

Hoh, Ramona A.; Stowe, Timothy R.; Turk, Erin; Stearns, Tim

2012-01-01

Defects in the centrosome and cilium are associated with a set of human diseases having diverse phenotypes. To further characterize the components that define the function of these organelles we determined the transcriptional profile of multiciliated tracheal epithelial cells. Cultures of mouse tracheal epithelial cells undergoing differentiation in vitro were derived from mice expressing GFP from the ciliated-cell specific FOXJ1 promoter (FOXJ1:GFP). The transcriptional profile of ciliating GFP+ cells from these cultures was defined at an early and a late time point during differentiation and was refined by subtraction of the profile of the non-ciliated GFP- cells. We identified 649 genes upregulated early, when most cells were forming basal bodies, and 73 genes genes upregulated late, when most cells were fully ciliated. Most, but not all, of known centrosome proteins are transcriptionally upregulated early, particularly Plk4, a master regulator of centriole formation. We found that three genes associated with human disease states, Mdm1, Mlf1, and Dyx1c1, are upregulated during ciliogenesis and localize to centrioles and cilia. This transcriptome for mammalian multiciliated epithelial cells identifies new candidate centrosome and cilia proteins, highlights similarities between components of motile and primary cilia, and identifies new links between cilia proteins and human disease. PMID:23300604

Kif3a Controls Murine Nephron Number Via GLI3 Repressor, Cell Survival, and Gene Expression in a Lineage-Specific Manner

PubMed Central

Chi, Lijun; Galtseva, Alevtina; Chen, Lin; Mo, Rong; Hui, Chi-chung; Rosenblum, Norman D.

2013-01-01

The primary cilium is required during early embryo patterning, epithelial tubulogenesis, and growth factor-dependent signal transduction. The requirement for primary cilia during renal epithelial-mesenchymal tissue interactions that give rise to nephrons is undefined. Here, we used Cre-mediated recombination to generate mice with Kif3a deficiency targeted to the ureteric and/or metanephric mesenchyme cell lineages in the embryonic kidney. Gradual loss of primary cilia in either lineage leads to a phenotype of reduced nephron number. Remarkably, in addition to cyst formation, loss of primary cilia in the ureteric epithelial cell leads to decreased expression of Wnt11 and Ret and reduced ureteric branching. Constitutive expression of GLI3 repressor (Gli3Δ699/+) rescues these abnormalities. In embryonic metanephric mesenchyme cells, Kif3a deficiency limits survival of nephrogenic progenitor cells and expression of genes required for nephron formation. Together, our data demonstrate that Kif3a controls nephron number via distinct cell lineage-specific mechanisms. PMID:23762375

Ciliary dysfunction and obesity.

PubMed

Mok, C A; Héon, E; Zhen, M

2010-01-01

Obesity associates with increased health risks such as heart disease, stroke and diabetes. The steady rise in the obese population worldwide poses an increasing burden on health systems. Genetic factors contribute to the development of obesity, and the elucidation of their physiological functions helps to understand the cause, and improve the prevention, diagnosis and treatment for this disorder. Primary cilia are evolutionarily conserved organelles whose dysfunctions lead to human disorders now defined as ciliopathies. Human ciliopathies present pleiotropic and overlapping phenotypes that often include retinal degeneration, cystic renal anomalies and obesity. Increasing evidence implicates an intriguing involvement of cilia in lipid/energy homeostasis. Here we discuss recent studies in support of the key roles of ciliary genes in the development and pathology of obesity in various animal models. Genes affecting ciliary development and function may pose promising candidate underlying genetic factors that contribute to the development of common obesity.

Magnetically Actuated Cilia for Microfluidic Manipulation

NASA Astrophysics Data System (ADS)

Hanasoge, Srinivas; Owen, Drew; Ballard, Matt; Hesketh, Peter J.; Alexeev, Alexander; Woodruff School of Mechanical Engineering Collaboration; Petit InstituteBioengineering; Biosciences Collaboration

2015-11-01

We demonstrate magnetic micro-cilia based microfluidic mixing and capture techniques. For this, we use a simple and easy to fabricate high aspect ratio cilia, which are actuated magnetically. These micro-features are fabricated by evaporating NiFe alloy at room temperature, on to patterned photoresist. The evaporated alloy curls upwards when the seed layer is removed to release the cilia, thus making a free standing `C' shaped magnetic microstructure. This is actuated using an external electromagnet or a rotating magnet. The artificial cilia can be actuated upto 20Hz. We demonstrate the active mixing these cilia can produce in the microchannel. Also, we demonstrate the capture of target species in a sample using these fast oscillating cilia. The surface of the cilia is functionalized by streptavidin which binds to biotin labelled fluorescent microspheres and mimic the capture of bacteria. We show very high capture efficiencies by using these methods. These simple to fabricate micro cilia can easily be incorporated into many microfluidic systems which require high mixing and capture efficiencies.

Primary Cilia Negatively Regulate Melanogenesis in Melanocytes and Pigmentation in a Human Skin Model.

PubMed

Choi, Hyunjung; Shin, Ji Hyun; Kim, Eun Sung; Park, So Jung; Bae, Il-Hong; Jo, Yoon Kyung; Jeong, In Young; Kim, Hyoung-June; Lee, Youngjin; Park, Hea Chul; Jeon, Hong Bae; Kim, Ki Woo; Lee, Tae Ryong; Cho, Dong-Hyung

2016-01-01

The primary cilium is an organelle protruding from the cell body that senses external stimuli including chemical, mechanical, light, osmotic, fluid flow, and gravitational signals. Skin is always exposed to the external environment and responds to external stimuli. Therefore, it is possible that primary cilia have an important role in skin. Ciliogenesis was reported to be involved in developmental processes in skin, such as keratinocyte differentiation and hair formation. However, the relation between skin pigmentation and primary cilia is largely unknown. Here, we observed that increased melanogenesis in melanocytes treated with a melanogenic inducer was inhibited by a ciliogenesis inducer, cytochalasin D, and serum-free culture. However, these inhibitory effects disappeared in GLI2 knockdown cells. In addition, activation of sonic hedgehog (SHH)-smoothened (Smo) signaling pathway by a Smo agonist, SAG inhibited melanin synthesis in melanocytes and pigmentation in a human skin model. On the contrary, an inhibitor of primary cilium formation, ciliobrevin A1, activated melanogenesis in melanocytes. These results suggest that skin pigmentation may be regulated partly by the induction of ciliogenesis through Smo-GLI2 signaling.

Intestinal cell kinase, a protein associated with endocrine-cerebro-osteodysplasia syndrome, is a key regulator of cilia length and Hedgehog signaling.

PubMed

Moon, Heejung; Song, Jieun; Shin, Jeong-Oh; Lee, Hankyu; Kim, Hong-Kyung; Eggenschwiller, Jonathan T; Bok, Jinwoong; Ko, Hyuk Wan

2014-06-10

Endocrine-cerebro-osteodysplasia (ECO) syndrome is a recessive genetic disorder associated with multiple congenital defects in endocrine, cerebral, and skeletal systems that is caused by a missense mutation in the mitogen-activated protein kinase-like intestinal cell kinase (ICK) gene. In algae and invertebrates, ICK homologs are involved in flagellar formation and ciliogenesis, respectively. However, it is not clear whether this role of ICK is conserved in mammals and how a lack of functional ICK results in the characteristic phenotypes of human ECO syndrome. Here, we generated Ick knockout mice to elucidate the precise role of ICK in mammalian development and to examine the pathological mechanisms of ECO syndrome. Ick null mouse embryos displayed cleft palate, hydrocephalus, polydactyly, and delayed skeletal development, closely resembling ECO syndrome phenotypes. In cultured cells, down-regulation of Ick or overexpression of kinase-dead or ECO syndrome mutant ICK resulted in an elongation of primary cilia and abnormal Sonic hedgehog (Shh) signaling. Wild-type ICK proteins were generally localized in the proximal region of cilia near the basal bodies, whereas kinase-dead ICK mutant proteins accumulated in the distal part of bulged ciliary tips. Consistent with these observations in cultured cells, Ick knockout mouse embryos displayed elongated cilia and reduced Shh signaling during limb digit patterning. Taken together, these results indicate that ICK plays a crucial role in controlling ciliary length and that ciliary defects caused by a lack of functional ICK leads to abnormal Shh signaling, resulting in congenital disorders such as ECO syndrome.

Branchial Cilia and Sperm Flagella Recruit Distinct Axonemal Components

PubMed Central

Konno, Alu; Shiba, Kogiku; Cai, Chunhua; Inaba, Kazuo

2015-01-01

Eukaryotic cilia and flagella have highly conserved 9 + 2 structures. They are functionally diverged to play cell-type-specific roles even in a multicellular organism. Although their structural components are therefore believed to be common, few studies have investigated the molecular diversity of the protein components of the cilia and flagella in a single organism. Here we carried out a proteomic analysis and compared protein components between branchial cilia and sperm flagella in a marine invertebrate chordate, Ciona intestinalis. Distinct feature of protein recruitment in branchial cilia and sperm flagella has been clarified; (1) Isoforms of α- and β-tubulins as well as those of actins are distinctly used in branchial cilia or sperm flagella. (2) Structural components, such as dynein docking complex, tektins and an outer dense fiber protein, are used differently by the cilia and flagella. (3) Sperm flagella are specialized for the cAMP- and Ca2+-dependent regulation of outer arm dynein and for energy metabolism by glycolytic enzymes. Our present study clearly demonstrates that flagellar or ciliary proteins are properly recruited according to their function and stability, despite their apparent structural resemblance and conservation. PMID:25962172

The Arf GEF GBF1 and Arf4 synergize with the sensory receptor cargo, rhodopsin, to regulate ciliary membrane trafficking.

PubMed

Wang, Jing; Fresquez, Theresa; Kandachar, Vasundhara; Deretic, Dusanka

2017-12-01

The small GTPase Arf4 and the Arf GTPase-activating protein (GAP) ASAP1 cooperatively sequester sensory receptor cargo into transport carriers targeted to primary cilia, but the input that drives Arf4 activation in this process remains unknown. Here, we show, by using frog retinas and recombinant human proteins, that during the carrier biogenesis from the photoreceptor Golgi/ trans -Golgi network (TGN) a functional complex is formed between Arf4, the Arf guanine nucleotide exchange factor (GEF) GBF1 and the light-sensing receptor, rhodopsin. Rhodopsin and Arf4 bind the regulatory N-terminal dimerization and cyclophillin-binding (DCB)-homology upstream of Sec7 (HUS) domain of GBF1. The complex is sensitive to Golgicide A (GCA), a selective inhibitor of GBF1 that accordingly blocks rhodopsin delivery to the cilia, without disrupting the photoreceptor Golgi. The emergence of newly synthesized rhodopsin in the endomembrane system is essential for GBF1-Arf4 complex formation in vivo Notably, GBF1 interacts with the Arf GAP ASAP1 in a GCA-resistant manner. Our findings indicate that converging signals on GBF1 from the influx of cargo into the Golgi/TGN and the feedback from Arf4, combined with input from ASAP1, control Arf4 activation during sensory membrane trafficking to primary cilia. © 2017. Published by The Company of Biologists Ltd.

Caenorhabditis elegans as a model to study renal development and disease: sexy cilia.

PubMed

Barr, Maureen M

2005-02-01

The nematode Caenorhabditis elegans has no kidney per se, yet "the worm" has proved to be an excellent model to study renal-related issues, including tubulogenesis of the excretory canal, membrane transport and ion channel function, and human genetic diseases including autosomal dominant polycystic kidney disease (ADPKD). The goal of this review is to explain how C. elegans has provided insight into cilia development, cilia function, and human cystic kidney diseases.

Genome-wide screen identifies novel machineries required for both ciliogenesis and cell cycle arrest upon serum starvation

PubMed Central

Kim, Ji Hyun; Ki, Soo Mi; Joung, Je-Gun; Scott, Eric; Heynen-Genel, Susanne; Aza-Blanc, Pedro; Kwon, Chang Hyuk; Kim, Joon; Gleeson, Joseph G.; Lee, Ji Eun

2016-01-01

Biogenesis of the primary cilium, a cellular organelle mediating various signaling pathways, is generally coordinated with cell cycle exit/re-entry. Although the dynamic cell cycle-associated profile of the primary cilium has been largely accepted, the mechanism governing the link between ciliogenesis and cell cycle progression has been poorly understood. Using a human genome-wide RNAi screen, we identify genes encoding subunits of the spliceosome and proteasome as novel regulators of ciliogenesis. We demonstrate that 1) the mRNA processing-related hits are essential for RNA expression of molecules acting in cilia disassembly, such as AURKA and PLK1, and 2) the ubiquitin-proteasome systems (UPS)-involved hits are necessary for proteolysis of molecules acting in cilia assembly, such as IFT88 and CPAP. In particular, we show that these screen hit-associated mechanisms are crucial for both cilia assembly and cell cycle arrest in response to serum withdrawal. Finally, our data suggest that the mRNA processing mechanism may modulate the UPS-dependent decay of cilia assembly regulators to control ciliary resorption-coupled cell cycle re-entry. PMID:27033521

Left-right asymmetry: cilia stir up new surprises in the node.

PubMed

Babu, Deepak; Roy, Sudipto

2013-05-29

Cilia are microtubule-based hair-like organelles that project from the surface of most eukaryotic cells. They play critical roles in cellular motility, fluid transport and a variety of signal transduction pathways. While we have a good appreciation of the mechanisms of ciliary biogenesis and the details of their structure, many of their functions demand a more lucid understanding. One such function, which remains as intriguing as the time when it was first discovered, is how beating cilia in the node drive the establishment of left-right asymmetry in the vertebrate embryo. The bone of contention has been the two schools of thought that have been put forth to explain this phenomenon. While the 'morphogen hypothesis' believes that ciliary motility is responsible for the transport of a morphogen preferentially to the left side, the 'two-cilia model' posits that the motile cilia generate a leftward-directed fluid flow that is somehow sensed by the immotile sensory cilia on the periphery of the node. Recent studies with the mouse embryo argue in favour of the latter scenario. Yet this principle may not be generally conserved in other vertebrates that use nodal flow to specify their left-right axis. Work with the teleost fish medaka raises the tantalizing possibility that motility as well as sensory functions of the nodal cilia could be residing within the same organelle. In the end, how ciliary signalling is transmitted to institute asymmetric gene expression that ultimately induces asymmetric organogenesis remains unresolved.

Left–right asymmetry: cilia stir up new surprises in the node

PubMed Central

Babu, Deepak; Roy, Sudipto

2013-01-01

Cilia are microtubule-based hair-like organelles that project from the surface of most eukaryotic cells. They play critical roles in cellular motility, fluid transport and a variety of signal transduction pathways. While we have a good appreciation of the mechanisms of ciliary biogenesis and the details of their structure, many of their functions demand a more lucid understanding. One such function, which remains as intriguing as the time when it was first discovered, is how beating cilia in the node drive the establishment of left–right asymmetry in the vertebrate embryo. The bone of contention has been the two schools of thought that have been put forth to explain this phenomenon. While the ‘morphogen hypothesis’ believes that ciliary motility is responsible for the transport of a morphogen preferentially to the left side, the ‘two-cilia model’ posits that the motile cilia generate a leftward-directed fluid flow that is somehow sensed by the immotile sensory cilia on the periphery of the node. Recent studies with the mouse embryo argue in favour of the latter scenario. Yet this principle may not be generally conserved in other vertebrates that use nodal flow to specify their left–right axis. Work with the teleost fish medaka raises the tantalizing possibility that motility as well as sensory functions of the nodal cilia could be residing within the same organelle. In the end, how ciliary signalling is transmitted to institute asymmetric gene expression that ultimately induces asymmetric organogenesis remains unresolved. PMID:23720541

Ciliary proteins Bbs8 and Ift20 promote planar cell polarity in the cochlea

PubMed Central

May-Simera, Helen L.; Petralia, Ronald S.; Montcouquiol, Mireille; Wang, Ya-Xian; Szarama, Katherine B.; Liu, Yun; Lin, Weichun; Deans, Michael R.; Pazour, Gregory J.; Kelley, Matthew W.

2015-01-01

Primary cilia have been implicated in the generation of planar cell polarity (PCP). However, variations in the severity of polarity defects in different cilia mutants, coupled with recent demonstrations of non-cilia-related actions of some cilia genes, make it difficult to determine the basis of these polarity defects. To address this issue, we evaluated PCP defects in cochlea from a selection of mice with mutations in cilia-related genes. Results indicated notable PCP defects, including mis-oriented hair cell stereociliary bundles, in Bbs8 and Ift20 single mutants that are more severe than in other cilia gene knockouts. In addition, deletion of either Bbs8 or Ift20 results in disruptions in asymmetric accumulation of the core PCP molecule Vangl2 in cochlear cells, suggesting a role for Bbs8 and/or Ift20, possibly upstream of core PCP asymmetry. Consistent with this, co-immunoprecipitation experiments indicate direct interactions of Bbs8 and Ift20 with Vangl2. We observed localization of Bbs and Ift proteins to filamentous actin as well as microtubules. This could implicate these molecules in selective trafficking of membrane proteins upstream of cytoskeletal reorganization, and identifies new roles for cilia-related proteins in cochlear PCP. PMID:25605782

Cilia in vertebrate left–right patterning

PubMed Central

Dasgupta, Agnik

2016-01-01

Understanding how left–right (LR) asymmetry is generated in vertebrate embryos is an important problem in developmental biology. In humans, a failure to align the left and right sides of cardiovascular and/or gastrointestinal systems often results in birth defects. Evidence from patients and animal models has implicated cilia in the process of left–right patterning. Here, we review the proposed functions for cilia in establishing LR asymmetry, which include creating transient leftward fluid flows in an embryonic ‘left–right organizer’. These flows direct asymmetric activation of a conserved Nodal (TGFβ) signalling pathway that guides asymmetric morphogenesis of developing organs. We discuss the leading hypotheses for how cilia-generated asymmetric fluid flows are translated into asymmetric molecular signals. We also discuss emerging mechanisms that control the subcellular positioning of cilia and the cellular architecture of the left–right organizer, both of which are critical for effective cilia function during left–right patterning. Finally, using mosaic cell-labelling and time-lapse imaging in the zebrafish embryo, we provide new evidence that precursor cells maintain their relative positions as they give rise to the ciliated left–right organizer. This suggests the possibility that these cells acquire left–right positional information prior to the appearance of cilia. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’. PMID:27821522

Cilia in vertebrate left-right patterning.

PubMed

Dasgupta, Agnik; Amack, Jeffrey D

2016-12-19

Understanding how left-right (LR) asymmetry is generated in vertebrate embryos is an important problem in developmental biology. In humans, a failure to align the left and right sides of cardiovascular and/or gastrointestinal systems often results in birth defects. Evidence from patients and animal models has implicated cilia in the process of left-right patterning. Here, we review the proposed functions for cilia in establishing LR asymmetry, which include creating transient leftward fluid flows in an embryonic 'left-right organizer'. These flows direct asymmetric activation of a conserved Nodal (TGFβ) signalling pathway that guides asymmetric morphogenesis of developing organs. We discuss the leading hypotheses for how cilia-generated asymmetric fluid flows are translated into asymmetric molecular signals. We also discuss emerging mechanisms that control the subcellular positioning of cilia and the cellular architecture of the left-right organizer, both of which are critical for effective cilia function during left-right patterning. Finally, using mosaic cell-labelling and time-lapse imaging in the zebrafish embryo, we provide new evidence that precursor cells maintain their relative positions as they give rise to the ciliated left-right organizer. This suggests the possibility that these cells acquire left-right positional information prior to the appearance of cilia.This article is part of the themed issue 'Provocative questions in left-right asymmetry'. © 2016 The Author(s).

Mutation of the MAP kinase DYF-5 affects docking and undocking of kinesin-2 motors and reduces their speed in the cilia of Caenorhabditis elegans

PubMed Central

Burghoorn, Jan; Dekkers, Martijn P. J.; Rademakers, Suzanne; de Jong, Ton; Willemsen, Rob; Jansen, Gert

2007-01-01

In the cilia of the nematode Caenorhabditis elegans, anterograde intraflagellar transport (IFT) is mediated by two kinesin-2 complexes, kinesin II and OSM-3 kinesin. These complexes function together in the cilia middle segments, whereas OSM-3 alone mediates transport in the distal segments. Not much is known about the mechanisms that compartmentalize the kinesin-2 complexes or how transport by both kinesins is coordinated. Here, we identify DYF-5, a conserved MAP kinase that plays a role in these processes. Fluorescence microscopy and EM revealed that the cilia of dyf-5 loss-of-function (lf) animals are elongated and are not properly aligned into the amphid channel. Some cilia do enter the amphid channel, but the distal ends of these cilia show accumulation of proteins. Consistent with these observations, we found that six IFT proteins accumulate in the cilia of dyf-5(lf) mutants. In addition, using genetic analyses and live imaging to measure the motility of IFT proteins, we show that dyf-5 is required to restrict kinesin II to the cilia middle segments. Finally, we show that, in dyf-5(lf) mutants, OSM-3 moves at a reduced speed and is not attached to IFT particles. We propose that DYF-5 plays a role in the undocking of kinesin II from IFT particles and in the docking of OSM-3 onto IFT particles. PMID:17420466

Ciliary heterogeneity within a single cell: the Paramecium model.

PubMed

Aubusson-Fleury, Anne; Cohen, Jean; Lemullois, Michel

2015-01-01

Paramecium is a single cell able to divide in its morphologically differentiated stage that has many cilia anchored at its cell surface. Many thousands of cilia are thus assembled in a short period of time during division to duplicate the cell pattern while the cell continues swimming. Most, but not all, of these sensory cilia are motile and involved in two main functions: prey capture and cell locomotion. These cilia display heterogeneity, both in their length and their biochemical properties. Thanks to these properties, as well as to the availability of many postgenomic tools and the possibility to follow the regrowth of cilia after deciliation, Paramecium offers a nice opportunity to study the assembly of the cilia, as well as the genesis of their diversity within a single cell. In this paper, after a brief survey of Paramecium morphology and cilia properties, we describe the tools and the protocols currently used for immunofluorescence, transmission electron microscopy, and ultrastructural immunocytochemistry to analyze cilia, with special recommendations to overcome the problem raised by cilium diversity. Copyright © 2015. Published by Elsevier Inc.

STUDIES ON CILIA

PubMed Central

Satir, Peter

1963-01-01

Upon excision into spring water, the lateral cilia of the gill of the freshwater mussel Elliptio complanatus (Solander) stop beating, but 0.04 M potassium ion can activate the gill so that these cilia again beat with metachronal rhythm. One per cent osmium tetroxide quickly pipetted onto a fully activated gill fixes the lateral cilia in a pattern that preserves the form and arrangement of the metachronal wave, and permits the cilia to be studied with the electron microscope in all stages of their beat cycle. Changes are seen in the fixed active preparation that are not present in the inactive control, i.e., in the packing of the cilia, the position of the axis of the ciliary cross-section, and the diameter of the ring of peripheral filaments. Analysis of these parameters may lead to new correlations between ciliary fine structure and function. PMID:14079494

Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways

PubMed Central

Geneva, Ivayla I.; Tan, Han Yen; Calvert, Peter D.

2017-01-01

Resolution limitations of optical systems are major obstacles for determining whether proteins are enriched within cell compartments. Here we use an approach to determine the degree of membrane protein ciliary enrichment that quantitatively accounts for the differences in sampling of the ciliary and apical membranes inherent to confocal microscopes. Theory shows that cilia will appear more than threefold brighter than the surrounding apical membrane when the densities of fluorescently labeled proteins are the same, thus providing a benchmark for ciliary enrichment. Using this benchmark, we examined the ciliary enrichment signals of two G protein–coupled receptors (GPCRs)—the somatostatin receptor 3 and rhodopsin. Remarkably, we found that the C-terminal VxPx motif, required for efficient enrichment of rhodopsin within rod photoreceptor sensory cilia, inhibited enrichment of the somatostatin receptor in primary cilia. Similarly, VxPx inhibited primary cilium enrichment of a chimera of rhodopsin and somatostatin receptor 3, where the dual Ax(S/A)xQ ciliary targeting motifs within the third intracellular loop of the somatostatin receptor replaced the third intracellular loop of rhodopsin. Rhodopsin was depleted from primary cilia but gained access, without being enriched, with the dual Ax(S/A)xQ motifs. Ciliary enrichment of these GPCRs thus operates via distinct mechanisms in different cells. PMID:27974638

A sliding-control switch stabilizes synchronized states in a model of actuated cilia

NASA Astrophysics Data System (ADS)

Buchmann, Amy; Cortez, Ricardo; Fauci, Lisa

2017-11-01

A key function of cilia, flexible hairlike appendages located on the surface of a cell, is the transport of mucus in the lungs, where the cilia self-organize forming a metachronal wave that propels the surrounding fluid. Cilia also play an important role in the locomotion of ciliated microswimmers and other biological processes. To analyze the coordinated movement of cilia interacting through a fluid, we model each cilium as an elastic, actuated body whose beat pattern is driven by a geometric switch that drives the motion of the power and recovery strokes. The cilia are coupled to the viscous fluid using a numerical method based upon a centerline distribution of regularized Stokeslets. We first characterize the beat cycle and flow produced by a single cilium and then present results on the synchronization states between two cilia that show that the in-phase equilibrium is unstable while the anti-phase equilibrium is stable under the geometric switch model. Adding a sliding-control switching mechanism stabilizes the in-phase motion.

Applications of emerging transmission electron microscopy technology in PCD research and diagnosis.

PubMed

Shoemark, Amelia

2017-01-01

Primary Ciliary Dyskinesia (PCD) is a heterogeneous genetic condition characterized by dysfunction of motile cilia. Patients suffer from chronic infection and inflammation of the upper and lower respiratory tract. Diagnosis of PCD is confirmed by identification of a hallmark defect of ciliary ultrastructure or by identification of biallelic pathogenic mutations in a known PCD gene. Since the first description of PCD in 1976, assessment of ciliary ultrastructure by transmission electron microscopy (TEM) has been central to diagnosis and research. Electron tomography is a technique whereby a series of transmission electron micrographs are collected at different angles and reconstructed into a single 3D model of a specimen. Electron tomography provides improved spatial information and resolution compared to a single micrograph. Research by electron tomography has revealed new insight into ciliary ultrastructure and consequently ciliary function at a molecular and cellular level. Gene discovery studies in PCD have utilized electron tomography to define the structural consequences of variants in cilia genes. Modern transmission electron microscopes capable of electron tomography are increasingly being installed in clinical laboratories. This presents the possibility for the use of tomography technique in a diagnostic setting. This review describes the electron tomography technique, the contribution tomography has made to the understanding of basic cilia structure and function and finally the potential of the technique for use in PCD diagnosis.

Primary ciliary dyskinesia: current state of the art

PubMed Central

Bush, Andrew; Chodhari, Rahul; Collins, Nicola; Copeland, Fiona; Hall, Pippa; Harcourt, Jonny; Hariri, Mohamed; Hogg, Claire; Lucas, Jane; Mitchison, Hannah M; O'Callaghan, Christopher; Phillips, Gill

2007-01-01

Primary ciliary dyskinesia (PCD) is usually inherited as an autosomal recessive disorder and presents with upper and lower respiratory tract infection, and mirror image arrangement in around 50% of cases. Cilia dysfunction is also implicated in a wider spectrum of disease, including polycystic liver and kidney disease, central nervous system problems including retinopathy and hydrocephalus, and biliary atresia. Cilia are complex structures, containing more than 250 proteins; recent studies have begun to locate PCD genes scattered throughout the genome. Screening tests for PCD include nasal nitric oxide and in vivo tests of ciliary motility such as the saccharin test. Specific diagnosis requires examination of cilia by light and electron microscopy, with epithelial culture in doubtful cases. This is only available in supra‐regional centres, recently centrally funded by the National Commissioning Group. Treatment is not evidence based and recommendations are largely extrapolated from cystic fibrosis and other suppurative lung diseases. PMID:17634184

Centrioles, centrosomes, and cilia in health and disease.

PubMed

Nigg, Erich A; Raff, Jordan W

2009-11-13

Centrioles are barrel-shaped structures that are essential for the formation of centrosomes, cilia, and flagella. Here we review recent advances in our understanding of the function and biogenesis of these organelles, and we emphasize their connection to human disease. Deregulation of centrosome numbers has long been proposed to contribute to genome instability and tumor formation, whereas mutations in centrosomal proteins have recently been genetically linked to microcephaly and dwarfism. Finally, structural or functional centriole aberrations contribute to ciliopathies, a variety of complex diseases that stem from the absence or dysfunction of cilia.

Mice Deficient in the Axonemal Protein Tektin-t Exhibit Male Infertility and Immotile-Cilium Syndrome Due to Impaired Inner Arm Dynein Function

PubMed Central

Tanaka, Hiromitsu; Iguchi, Naoko; Toyama, Yoshiro; Kitamura, Kouichi; Takahashi, Tohru; Kaseda, Kazuhiro; Maekawa, Mamiko; Nishimune, Yoshitake

2004-01-01

The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia. PMID:15340058

The sense of smell, its signalling pathways, and the dichotomy of cilia and microvilli in olfactory sensory cells

PubMed Central

2007-01-01

Smell is often regarded as an ancillary perception in primates, who seem so dominated by their sense of vision. In this paper, we will portray some aspects of the significance of olfaction to human life and speculate on what evolutionary factors contribute to keeping it alive. We then outline the functional architecture of olfactory sensory neurons and their signal transduction pathways, which are the primary detectors that render olfactory perception possible. Throughout the phylogenetic tree, olfactory neurons, at their apical tip, are either decorated with cilia or with microvilli. The significance of this dichotomy is unknown. It is generally assumed that mammalian olfactory neurons are of the ciliary type only. The existance of so-called olfactory microvillar cells in mammals, however, is well documented, but their nature remains unclear and their function orphaned. This paper discusses the possibility, that in the main olfactory epithelium of mammals ciliated and microvillar sensory cells exist concurrently. We review evidence related to this hypothesis and ask, what function olfactory microvillar cells might have and what signalling mechanisms they use. PMID:17903277

Mutations in RSPH1 cause primary ciliary dyskinesia with a unique clinical and ciliary phenotype.

PubMed

Knowles, Michael R; Ostrowski, Lawrence E; Leigh, Margaret W; Sears, Patrick R; Davis, Stephanie D; Wolf, Whitney E; Hazucha, Milan J; Carson, Johnny L; Olivier, Kenneth N; Sagel, Scott D; Rosenfeld, Margaret; Ferkol, Thomas W; Dell, Sharon D; Milla, Carlos E; Randell, Scott H; Yin, Weining; Sannuti, Aruna; Metjian, Hilda M; Noone, Peadar G; Noone, Peter J; Olson, Christina A; Patrone, Michael V; Dang, Hong; Lee, Hye-Seung; Hurd, Toby W; Gee, Heon Yung; Otto, Edgar A; Halbritter, Jan; Kohl, Stefan; Kircher, Martin; Krischer, Jeffrey; Bamshad, Michael J; Nickerson, Deborah A; Hildebrandt, Friedhelm; Shendure, Jay; Zariwala, Maimoona A

2014-03-15

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia, but the genetic cause is not defined for all patients with PCD. To identify disease-causing mutations in novel genes, we performed exome sequencing, follow-up characterization, mutation scanning, and genotype-phenotype studies in patients with PCD. Whole-exome sequencing was performed using NimbleGen capture and Illumina HiSeq sequencing. Sanger-based sequencing was used for mutation scanning, validation, and segregation analysis. We performed exome sequencing on an affected sib-pair with normal ultrastructure in more than 85% of cilia. A homozygous splice-site mutation was detected in RSPH1 in both siblings; parents were carriers. Screening RSPH1 in 413 unrelated probands, including 325 with PCD and 88 with idiopathic bronchiectasis, revealed biallelic loss-of-function mutations in nine additional probands. Five affected siblings of probands in RSPH1 families harbored the familial mutations. The 16 individuals with RSPH1 mutations had some features of PCD; however, nasal nitric oxide levels were higher than in patients with PCD with other gene mutations (98.3 vs. 20.7 nl/min; P < 0.0003). Additionally, individuals with RSPH1 mutations had a lower prevalence (8 of 16) of neonatal respiratory distress, and later onset of daily wet cough than typical for PCD, and better lung function (FEV1), compared with 75 age- and sex-matched PCD cases (73.0 vs. 61.8, FEV1 % predicted; P = 0.043). Cilia from individuals with RSPH1 mutations had normal beat frequency (6.1 ± Hz at 25°C), but an abnormal, circular beat pattern. The milder clinical disease and higher nasal nitric oxide in individuals with biallelic mutations in RSPH1 provides evidence of a unique genotype-phenotype relationship in PCD, and suggests that mutations in RSPH1 may be associated with residual ciliary function.

Deciliation Is Associated with Dramatic Remodeling of Epithelial Cell Junctions and Surface Domains

PubMed Central

Overgaard, Christian E.; Sanzone, Kaitlin M.; Spiczka, Krystle S.; Sheff, David R.; Sandra, Alexander

2009-01-01

Stress-induced shedding of motile cilia (autotomy) has been documented in diverse organisms and likely represents a conserved cellular reaction. However, little is known about whether primary cilia are shed from mammalian epithelial cells and what impact deciliation has on polarized cellular organization. We show that several chemically distinct agents trigger autotomy in epithelial cells. Surprisingly, deciliation is associated with a significant, but reversible increase in transepithelial resistance. This reflects substantial reductions in tight junction proteins associated with “leaky” nephron segments (e.g., claudin-2). At the same time, apical trafficking of gp80/clusterin and gp114/CEACAM becomes randomized, basal-lateral delivery of Na,K-ATPase is reduced, and expression of the nonciliary apical protein gp135/podocalyxin is greatly decreased. However, ciliogenesis-impaired MDCK cells do not undergo continual junction remodeling, and mature cilia are not required for autotomy-associated remodeling events. Deciliation and epithelial remodeling may be mechanistically linked processes, because RNAi-mediated reduction of Exocyst subunit Sec6 inhibits ciliary shedding and specifically blocks deciliation-associated down-regulation of claudin-2 and gp135. We propose that ciliary autotomy represents a signaling pathway that impacts the organization and function of polarized epithelial cells. PMID:19005211

Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins

PubMed Central

Shimada, Issei S.; Loriot, Evan

2017-01-01

The primary cilium is a paradigmatic organelle for studying compartmentalized signaling; however, unlike soluble protein trafficking, processes targeting integral membrane proteins to cilia are poorly understood. In this study, we determine that the tubby family protein TULP3 functions as a general adapter for ciliary trafficking of structurally diverse integral membrane cargo, including multiple reported and novel rhodopsin family G protein–coupled receptors (GPCRs) and the polycystic kidney disease–causing polycystin 1/2 complex. The founding tubby family member TUB also localizes to cilia similar to TULP3 and determines trafficking of a subset of these GPCRs to neuronal cilia. Using minimal ciliary localization sequences from GPCRs and fibrocystin (also implicated in polycystic kidney disease), we demonstrate these motifs to be sufficient and TULP3 dependent for ciliary trafficking. We propose a three-step model for TULP3/TUB-mediated ciliary trafficking, including the capture of diverse membrane cargo by the tubby domain in a phosphoinositide 4,5-bisphosphate (PI(4,5)P2)-dependent manner, ciliary delivery by intraflagellar transport complex A binding to the TULP3/TUB N terminus, and subsequent release into PI(4,5)P2-deficient ciliary membrane. PMID:28154160

Ndel1 suppresses ciliogenesis in proliferating cells by regulating the trichoplein-Aurora A pathway.

PubMed

Inaba, Hironori; Goto, Hidemasa; Kasahara, Kousuke; Kumamoto, Kanako; Yonemura, Shigenobu; Inoko, Akihito; Yamano, Shotaro; Wanibuchi, Hideki; He, Dongwei; Goshima, Naoki; Kiyono, Tohru; Hirotsune, Shinji; Inagaki, Masaki

2016-02-15

Primary cilia protrude from the surface of quiescent cells and disassemble at cell cycle reentry. We previously showed that ciliary reassembly is suppressed by trichoplein-mediated Aurora A activation pathway in growing cells. Here, we report that Ndel1, a well-known modulator of dynein activity, localizes at the subdistal appendage of the mother centriole, which nucleates a primary cilium. In the presence of serum, Ndel1 depletion reduces trichoplein at the mother centriole and induces unscheduled primary cilia formation, which is reverted by forced trichoplein expression or coknockdown of KCTD17 (an E3 ligase component protein for trichoplein). Serum starvation induced transient Ndel1 degradation, subsequent to the disappearance of trichoplein at the mother centriole. Forced expression of Ndel1 suppressed trichoplein degradation and axonemal microtubule extension during ciliogenesis, similar to trichoplein induction or KCTD17 knockdown. Most importantly, the proportion of ciliated and quiescent cells was increased in the kidney tubular epithelia of newborn Ndel1-hypomorphic mice. Thus, Ndel1 acts as a novel upstream regulator of the trichoplein-Aurora A pathway to inhibit primary cilia assembly. © 2016 Inaba et al.

Cell- and subunit-specific mechanisms of CNG channel ciliary trafficking and localization in C. elegans

PubMed Central

Wojtyniak, Martin; Brear, Andrea G.; O'Halloran, Damien M.; Sengupta, Piali

2013-01-01

Summary Primary cilia are ubiquitous sensory organelles that concentrate transmembrane signaling proteins essential for sensing environmental cues. Mislocalization of crucial ciliary signaling proteins, such as the tetrameric cyclic nucleotide-gated (CNG) channels, can lead to cellular dysfunction and disease. Although several cis- and trans-acting factors required for ciliary protein trafficking and localization have been identified, whether these mechanisms act in a protein- and cell-specific manner is largely unknown. Here, we show that CNG channel subunits can be localized to discrete ciliary compartments in individual sensory neurons in C. elegans, suggesting that channel composition is heterogeneous across the cilium. We demonstrate that ciliary localization of CNG channel subunits is interdependent on different channel subunits in specific cells, and identify sequences required for efficient ciliary targeting and localization of the TAX-2 CNGB and TAX-4 CNGA subunits. Using a candidate gene approach, we show that Inversin, transition zone proteins, intraflagellar transport motors and a MYND-domain protein are required to traffic and/or localize CNG channel subunits in both a cell- and channel subunit-specific manner. We further find that TAX-2 and TAX-4 are relatively immobile in specific sensory cilia subcompartments, suggesting that these proteins undergo minimal turnover in these domains in mature cilia. Our results uncover unexpected diversity in the mechanisms that traffic and localize CNG channel subunits to cilia both within and across cell types, highlighting the essential contribution of this process to cellular functions. PMID:23886944

Cell- and subunit-specific mechanisms of CNG channel ciliary trafficking and localization in C. elegans.

PubMed

Wojtyniak, Martin; Brear, Andrea G; O'Halloran, Damien M; Sengupta, Piali

2013-10-01

Primary cilia are ubiquitous sensory organelles that concentrate transmembrane signaling proteins essential for sensing environmental cues. Mislocalization of crucial ciliary signaling proteins, such as the tetrameric cyclic nucleotide-gated (CNG) channels, can lead to cellular dysfunction and disease. Although several cis- and trans-acting factors required for ciliary protein trafficking and localization have been identified, whether these mechanisms act in a protein- and cell-specific manner is largely unknown. Here, we show that CNG channel subunits can be localized to discrete ciliary compartments in individual sensory neurons in C. elegans, suggesting that channel composition is heterogeneous across the cilium. We demonstrate that ciliary localization of CNG channel subunits is interdependent on different channel subunits in specific cells, and identify sequences required for efficient ciliary targeting and localization of the TAX-2 CNGB and TAX-4 CNGA subunits. Using a candidate gene approach, we show that Inversin, transition zone proteins, intraflagellar transport motors and a MYND-domain protein are required to traffic and/or localize CNG channel subunits in both a cell- and channel subunit-specific manner. We further find that TAX-2 and TAX-4 are relatively immobile in specific sensory cilia subcompartments, suggesting that these proteins undergo minimal turnover in these domains in mature cilia. Our results uncover unexpected diversity in the mechanisms that traffic and localize CNG channel subunits to cilia both within and across cell types, highlighting the essential contribution of this process to cellular functions.

Unique among ciliopathies: primary ciliary dyskinesia, a motile cilia disorder.

PubMed

Praveen, Kavita; Davis, Erica E; Katsanis, Nicholas

2015-01-01

Primary ciliary dyskinesia (PCD) is a ciliopathy, but represents the sole entity from this class of disorders that results from the dysfunction of motile cilia. Characterized by respiratory problems appearing in childhood, infertility, and situs defects in ~50% of individuals, PCD has an estimated prevalence of approximately 1 in 10,000 live births. The diagnosis of PCD can be prolonged due to a lack of disease awareness, coupled with the fact that symptoms can be confused with other more common genetic disorders, such as cystic fibrosis, or environmental insults that result in frequent respiratory infections. A primarily autosomal recessive disorder, PCD is genetically heterogeneous with >30 causal genes identified, posing significant challenges to genetic diagnosis. Here, we provide an overview of PCD as a disorder underscored by impaired ciliary motility; we discuss the recent advances towards uncovering the genetic basis of PCD; we discuss the molecular knowledge gained from PCD gene discovery, which has improved our understanding of motile ciliary assembly; and we speculate on how accelerated diagnosis, together with detailed phenotypic data, will shape the genetic and functional architecture of this disorder.

Variation in Cilia Protein Genes and Progression of Lung Disease in Cystic Fibrosis.

PubMed

Blue, Elizabeth; Louie, Tin L; Chong, Jessica X; Hebbring, Scott J; Barnes, Kathleen C; Rafaels, Nicholas M; Knowles, Michael R; Gibson, Ronald L; Bamshad, Michael J; Emond, Mary J

2018-04-01

Cystic fibrosis, like primary ciliary dyskinesia, is an autosomal recessive disorder characterized by abnormal mucociliary clearance and obstructive lung disease. We hypothesized that genes underlying the development or function of cilia may modify lung disease severity in persons with cystic fibrosis. To test this hypothesis, we compared variants in 93 candidate genes in both upper and lower tertiles of lung function in a large cohort of children and adults with cystic fibrosis with those of a population control dataset. Variants within candidate genes were tested for association using the SKAT-O test, comparing cystic fibrosis cases defined by poor (n = 127) or preserved (n = 127) lung function with population controls (n = 3,269 or 3,148, respectively). Associated variants were then tested for association with related phenotypes in independent datasets. Variants in DNAH14 and DNAAF3 were associated with poor lung function in cystic fibrosis, whereas variants in DNAH14 and DNAH6 were associated with preserved lung function in cystic fibrosis. Associations between DNAH14 and lung function were replicated in disease-related phenotypes characterized by obstructive lung disease in adults. Genetic variants within DNAH6, DNAH14, and DNAAF3 are associated with variation in lung function among persons with cystic fibrosis.

Exploring the Transcriptome of Ciliated Cells Using In Silico Dissection of Human Tissues

PubMed Central

Ivliev, Alexander E.; 't Hoen, Peter A. C.; van Roon-Mom, Willeke M. C.; Peters, Dorien J. M.; Sergeeva, Marina G.

2012-01-01

Cilia are cell organelles that play important roles in cell motility, sensory and developmental functions and are involved in a range of human diseases, known as ciliopathies. Here, we search for novel human genes related to cilia using a strategy that exploits the previously reported tendency of cell type-specific genes to be coexpressed in the transcriptome of complex tissues. Gene coexpression networks were constructed using the noise-resistant WGCNA algorithm in 12 publicly available microarray datasets from human tissues rich in motile cilia: airways, fallopian tubes and brain. A cilia-related coexpression module was detected in 10 out of the 12 datasets. A consensus analysis of this module's gene composition recapitulated 297 known and predicted 74 novel cilia-related genes. 82% of the novel candidates were supported by tissue-specificity expression data from GEO and/or proteomic data from the Human Protein Atlas. The novel findings included a set of genes (DCDC2, DYX1C1, KIAA0319) related to a neurological disease dyslexia suggesting their potential involvement in ciliary functions. Furthermore, we searched for differences in gene composition of the ciliary module between the tissues. A multidrug-and-toxin extrusion transporter MATE2 (SLC47A2) was found as a brain-specific central gene in the ciliary module. We confirm the localization of MATE2 in cilia by immunofluorescence staining using MDCK cells as a model. While MATE2 has previously gained attention as a pharmacologically relevant transporter, its potential relation to cilia is suggested for the first time. Taken together, our large-scale analysis of gene coexpression networks identifies novel genes related to human cell cilia. PMID:22558177

Integrin α8 and Pcdh15 act as a complex to regulate cilia biogenesis in sensory cells.

PubMed

Goodman, Linda; Zallocchi, Marisa

2017-11-01

The way an organism perceives its surroundings depends on sensory systems and the highly specialized cilia present in the neurosensory cells. Here, we describe the existence of an integrin α8 (Itga8) and protocadherin-15a (Pcdh15a) ciliary complex in neuromast hair cells in a zebrafish model. Depletion of the complex via downregulation or loss-of-function mutation leads to a dysregulation of cilia biogenesis and endocytosis. At the molecular level, removal of the complex blocks the access of Rab8a into the cilia as well as normal recruitment of ciliary cargo by centriolar satellites. These defects can be reversed by the introduction of a constitutively active form of Rhoa, suggesting that Itga8-Pcdh15a complex mediates its effect through the activation of this small GTPase and probably by the regulation of actin cytoskeleton dynamics. Our data points to a novel mechanism involved in the regulation of sensory cilia development, with the corresponding implications for normal sensory function. © 2017. Published by The Company of Biologists Ltd.

Clinical and Genetic Aspects of Primary Ciliary Dyskinesia / Kartagener Syndrome

PubMed Central

Leigh, Margaret W.; Pittman, Jessica E.; Carson, Johnny L.; Ferkol, Thomas W.; Dell, Sharon D.; Davis, Stephanie D.; Knowles, Michael R.; Zariwala, Maimoona A.

2013-01-01

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder of motile cilia. Most of the disease-causing mutations identified to date involve the heavy (DNAH5) or intermediate (DNAI1) chain dynein genes in ciliary outer dynein arms, although a few mutations have been noted in other genes. Clinical molecular genetic testing for PCD is available for the most common mutations. The respiratory manifestations of PCD (chronic bronchitis leading to bronchiectasis, chronic rhino-sinusitis and chronic otitis media) reflect impaired mucociliary clearance owing to defective axonemal structure. Ciliary ultrastructural analysis in most patients (>80%) reveals defective dynein arms, although defects in other axonemal components have also been observed. Approximately 50% of PCD patients have laterality defects (including situs inversus totalis and, less commonly, heterotaxy and congenital heart disease), reflecting dysfunction of embryological nodal cilia. Male infertility is common and reflects defects in sperm tail axonemes. Most PCD patients have a history of neonatal respiratory distress, suggesting that motile cilia play a role in fluid clearance during the transition from a fetal to neonatal lung. Ciliopathies involving sensory cilia, including autosomal dominant or recessive polycystic kidney disease, Bardet-Biedl syndrome, and Alstrom syndrome, may have chronic respiratory symptoms and even bronchiectasis suggesting clinical overlap with PCD. PMID:19606528

Primary cilia disruption differentially affects the infiltrating and resident macrophage compartment in the liver.

PubMed

Zimmerman, Kurt A; Song, Cheng Jack; Gonzalez-Mize, Nancy; Li, Zhang; Yoder, Bradley K

2018-06-01

Hepatorenal fibrocystic disease (HRFCD) is characterized by cysts in the kidney and liver with associated fibrosis and is the result of defects in proteins required for cilia function or assembly. Previous reports indicate that macrophages, mainly M2-like macrophages, contribute to HRFCD, although the origin of these cells (yolk sac-derived resident macrophages vs. bone marrow-derived infiltrating macrophages) and their contribution to the observed phenotypes are unknown. We utilize a congenital model of cilia dysfunction (IFT88 Orpk ) to study the importance of macrophages in HRFCD. Our data show a rapid expansion of the bile duct region and development of fibrosis between 2 and 4 wk of age. Immunofluorescence microscopy analysis reveals an accumulation of F4/80 + macrophages in regions exhibiting biliary hyperplasia in IFT88 Orpk mice. Flow cytometry data show that cilia dysfunction leads to an accumulation of infiltrating macrophages (CD11b hi , F4/80 lo ) and a reduction of resident macrophage (CD11b lo , F4/80 hi ) number. A majority of the infiltrating macrophages are Ly6c hi profibrogenic macrophages. Along with the accumulation of immune cells, expression of proinflammatory and profibrotic transcripts, including TGF-β, TNF-α, IL-1β, and chemokine (C-C) motif ligand 2, is increased. Quantitative RT-PCR analysis of flow-sorted cells shows enhanced expression of CCL2 in cholangiocytes and enhanced expression of VEGF-A and IL-6 in Ly6c hi macrophages. Genetic inhibition of Ly6c hi macrophage accumulation in IFT88 Orpk FVB CCR2 -/- mice reduced biliary fibrosis but did not affect epithelial expansion. Collectively, these studies suggest that biliary epithelium with defects in primary cilia preferentially recruits Ly6c hi infiltrating macrophages, which promote fibrotic progression in HRFCD pathogenesis. NEW & NOTEWORTHY These studies are the first to address the contribution of the infiltrating and resident macrophage niche during progression of hepatorenal fibrocystic disease (HRFCD). We show that the number of infiltrating macrophages is significantly upregulated in HRFCD mouse models. Finally, we show that prevention of Ly6c hi infiltrating macrophage accumulation significantly reduces biliary fibrosis, but not biliary hyperplasia, suggesting that this population may be responsible for the fibrotic progression of the disease in HRFCD patients.

Mutations in ZMYND10, a Gene Essential for Proper Axonemal Assembly of Inner and Outer Dynein Arms in Humans and Flies, Cause Primary Ciliary Dyskinesia

PubMed Central

Moore, Daniel J.; Onoufriadis, Alexandros; Shoemark, Amelia; Simpson, Michael A.; zur Lage, Petra I.; de Castro, Sandra C.; Bartoloni, Lucia; Gallone, Giuseppe; Petridi, Stavroula; Woollard, Wesley J.; Antony, Dinu; Schmidts, Miriam; Didonna, Teresa; Makrythanasis, Periklis; Bevillard, Jeremy; Mongan, Nigel P.; Djakow, Jana; Pals, Gerard; Lucas, Jane S.; Marthin, June K.; Nielsen, Kim G.; Santoni, Federico; Guipponi, Michel; Hogg, Claire; Antonarakis, Stylianos E.; Emes, Richard D.; Chung, Eddie M.K.; Greene, Nicholas D.E.; Blouin, Jean-Louis; Jarman, Andrew P.; Mitchison, Hannah M.

2013-01-01

Primary ciliary dyskinesia (PCD) is a ciliopathy characterized by airway disease, infertility, and laterality defects, often caused by dual loss of the inner dynein arms (IDAs) and outer dynein arms (ODAs), which power cilia and flagella beating. Using whole-exome and candidate-gene Sanger resequencing in PCD-affected families afflicted with combined IDA and ODA defects, we found that 6/38 (16%) carried biallelic mutations in the conserved zinc-finger gene BLU (ZMYND10). ZMYND10 mutations conferred dynein-arm loss seen at the ultrastructural and immunofluorescence level and complete cilia immotility, except in hypomorphic p.Val16Gly (c.47T>G) homozygote individuals, whose cilia retained a stiff and slowed beat. In mice, Zmynd10 mRNA is restricted to regions containing motile cilia. In a Drosophila model of PCD, Zmynd10 is exclusively expressed in cells with motile cilia: chordotonal sensory neurons and sperm. In these cells, P-element-mediated gene silencing caused IDA and ODA defects, proprioception deficits, and sterility due to immotile sperm. Drosophila Zmynd10 with an equivalent c.47T>G (p.Val16Gly) missense change rescued mutant male sterility less than the wild-type did. Tagged Drosophila ZMYND10 is localized primarily to the cytoplasm, and human ZMYND10 interacts with LRRC6, another cytoplasmically localized protein altered in PCD. Using a fly model of PCD, we conclude that ZMYND10 is a cytoplasmic protein required for IDA and ODA assembly and that its variants cause ciliary dysmotility and PCD with laterality defects. PMID:23891471

Smoothened-antagonists reverse homogentisic acid-induced alterations of Hedgehog signaling and primary cilium length in alkaptonuria.

PubMed

Gambassi, Silvia; Geminiani, Michela; Thorpe, Stephen D; Bernardini, Giulia; Millucci, Lia; Braconi, Daniela; Orlandini, Maurizio; Thompson, Clare L; Petricci, Elena; Manetti, Fabrizio; Taddei, Maurizio; Knight, Martin M; Santucci, Annalisa

2017-11-01

Alkaptonuria (AKU) is an ultra-rare genetic disease, in which the accumulation of a toxic metabolite, homogentisic acid (HGA) leads to the systemic development of ochronotic aggregates. These aggregates cause severe complications mainly at the level of joints with extensive degradation of the articular cartilage. Primary cilia have been demonstrated to play an essential role in development and the maintenance of articular cartilage homeostasis, through their involvement in mechanosignaling and Hedgehog signaling pathways. Hedgehog signaling has been demonstrated to be activated in osteoarthritis (OA) and to drive cartilage degeneration in vivo. The numerous similarities between OA and AKU suggest that primary cilia Hedgehog signaling may also be altered in AKU. Thus, we characterized an AKU cellular model in which healthy chondrocytes were treated with HGA (66 µM) to replicate AKU cartilage pathology. We investigated the degree of activation of the Hedgehog signaling pathway and how treatment with inhibitors of the receptor Smoothened (Smo) influenced Hedgehog activation and primary cilia structure. The results obtained in this work provide a further step in the comprehension of the pathophysiological features of AKU, suggesting a potential therapeutic approach to modulate AKU cartilage degradation processes through manipulation of the Hedgehog pathway. © 2016 Wiley Periodicals, Inc.

A primary cilia-dependent etiology for midline facial disorders

PubMed Central

Brugmann, Samantha A.; Allen, Nancy C.; James, Aaron W.; Mekonnen, Zesemayat; Madan, Elena; Helms, Jill A.

2010-01-01

Human faces exhibit enormous variation. When pathological conditions are superimposed on normal variation, a nearly unbroken series of facial morphologies is produced. When viewed in full, this spectrum ranges from cyclopia and hypotelorism to hypertelorism and facial duplications. Decreased Hedgehog pathway activity causes holoprosencephaly and hypotelorism. Here, we show that excessive Hedgehog activity, caused by truncating the primary cilia on cranial neural crest cells, causes hypertelorism and frontonasal dysplasia (FND). Elimination of the intraflagellar transport protein Kif3a leads to excessive Hedgehog responsiveness in facial mesenchyme, which is accompanied by broader expression domains of Gli1, Ptc and Shh, and reduced expression domains of Gli3. Furthermore, broader domains of Gli1 expression correspond to areas of enhanced neural crest cell proliferation in the facial prominences of Kif3a conditional knockouts. Avian Talpid embryos that lack primary cilia exhibit similar molecular changes and similar facial phenotypes. Collectively, these data support our hypothesis that a severe narrowing of the facial midline and excessive expansion of the facial midline are both attributable to disruptions in Hedgehog pathway activity. These data also raise the possibility that genes encoding ciliary proteins are candidates for human conditions of hypertelorism and FNDs. PMID:20106874

Imaging intraflagellar transport in mammalian primary cilia.

PubMed

Besschetnova, Tatiana Y; Roy, Barnali; Shah, Jagesh V

2009-01-01

The primary cilium is a specialized organelle that projects from the surface of many cell types. Unlike its motile counterpart it cannot beat but does transduce extracellular stimuli into intracellular signals and acts as a specialized subcellular compartment. The cilium is built and maintained by the transport of proteins and other biomolecules into and out of this compartment. The trafficking machinery for the cilium is referred to as IFT or intraflagellar transport. It was originally identified in the green algae Chlamydomonas and has been discovered throughout the evolutionary tree. The IFT machinery is widely conserved and acts to establish, maintain, and disassemble cilia and flagella. Understanding the role of IFT in cilium signaling and regulation requires a methodology for observing it directly. Here we describe current methods for observing the IFT process in mammalian primary cilia through the generation of fluorescent protein fusions and their expression in ciliated cell lines. The observation protocol uses high-resolution time-lapse microscopy to provide detailed quantitative measurements of IFT particle velocities in wild-type cells or in the context of genetic or other perturbations. Direct observation of IFT trafficking will provide a unique tool to dissect the processes that govern cilium regulation and signaling. 2009 Elsevier Inc. All rights reserved.

Centrosome Defects, Genetic Instability and Breast Cancer Progression

DTIC Science & Technology

2005-08-01

and is associated with cell cycle progression. Primary cilia loss is thought to be the cause of polycystic kidney disease, a condition in which kidney...polycyctin2 which is also implicated in polycystic kidney disease (Pazour, Baker et al. 2002; Pazour anrd Rosenbaum 2002; Pazour, San Agustin et al. 2002...1941-51. Pazour, G. J. (2004). "Intraflagellar transport and cilia-dependent renal disease: the ciliary hypothesis of polycystic kidney disease." J

The Roles of Primary Cilia in Cardiovascular System

DTIC Science & Technology

2016-10-01

mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen . Biochem J 134:707–716. Boveris A, Oshino N, Chance B. 1972...The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen . Biochem J 134:707–716. Boveris A, Oshino N...Aim 1.2 (months 13-30): We will examine signaling mechanisms of cilia & their effects on blood pressure. Aim 2 (months 7-36). We will study

Pix proteins and the evolution of centrioles.

PubMed

Woodland, Hugh R; Fry, Andrew M

2008-01-01

We have made a wide phylogenetic survey of Pix proteins, which are constituents of vertebrate centrioles in most eukaryotes. We have also surveyed the presence and structure of flagella or cilia and centrioles in these organisms, as far as is possible from published information. We find that Pix proteins are present in a vast range of eukaryotes, but not all. Where centrioles are absent so are Pix proteins. If one considers the maintenance of Pix proteins over evolutionary time scales, our analysis would suggest that their key function is to make cilia and flagella, and the same is true of centrioles. Moreover, this survey raises the possibility that Pix proteins are only maintained to make cilia and flagella that undulate, and even then only when they are constructed by transporting ciliary constituents up the cilium using the intraflagellar transport (IFT) system. We also find that Pix proteins have become generally divergent within Ecdysozoa and between this group and other taxa. This correlates with a simplification of centrioles within Ecdysozoa and a loss or divergence of cilia/flagella. Thus Pix proteins act as a weathervane to indicate changes in centriole function, whose core activity is to make cilia and flagella.

Generation of a three-dimensional ultrastructural model of human respiratory cilia.

PubMed

Burgoyne, Thomas; Dixon, Mellisa; Luther, Pradeep; Hogg, Claire; Shoemark, Amelia

2012-12-01

The ultrastructures of cilia and flagella are highly similar and well conserved through evolution. Consequently, Chlamydomonas is commonly used as a model organism for the study of human respiratory cilia. Since detailed models of Chlamydomonas axonemes were generated using cryoelectron tomography, disparities among some of the ultrastructural features have become apparent when compared with human cilia. Extrapolating information on human disease from the Chlamydomonas model may lead to discrepancies in translational research. This study aimed to establish the first three-dimensional ultrastructural model of human cilia. Tomograms of transverse sections (n = 6) and longitudinal sections (n = 9) of human nasal respiratory cilia were generated from three healthy volunteers. Key features of the cilium were resolved using subatomic averaging, and were measured. For validation of the method, a model of the well characterized structure of Chlamydomonas reinhardtii was simultaneously generated. Data were combined to create a fully quantified three-dimensional reconstruction of human nasal respiratory cilia. We highlight key differences in the axonemal sheath, microtubular doublets, radial spokes, and dynein arms between the two structures. We show a decreased axial periodicity of the radial spokes, inner dynein arms, and central pair protrusions in the human model. We propose that this first human model will provide a basis for research into the function and structure of human respiratory cilia in health and in disease.

A Novel Silicone-Magnetite Composite Material Used in the Fabrication of Biomimetic Cilia

NASA Astrophysics Data System (ADS)

Carstens, B. L.; Evans, B. A.; Shields, A. R.; Su, J.; Washburn, S.; Falvo, M. R.; Superfine, R.

2008-10-01

We have developed a novel polymer-magnetite composite that we use to fabricate arrays of magnetically actuable biomimetic cilia. Biomimetic cilia are flexible nanorods 750 nm in diameter and 25 microns tall. They generate fluid flows similar to those produced by biological cilia. Polymer-magnetic nanoparticle materials such as ours are becoming increasingly useful in biomedical applications and microelectromechanical systems (MEMS). Comprised of magnetite (Fe3O4), the nanoparticles have a diameter of 5-7 nm and are complexed with a silicone copolymer and crosslinked into a flexible, magnetic solid. Amine groups make up 6-7 percent of the silicone copolymer, providing a simple means of functionalization. We present a detailed mechanical and magnetic analysis of our bulk crosslinked material. The high-aspect ratio biomimetic cilia we create with this magnetite-copolymer complex may have applications in microfluidic mixing, biofouling, and MEMS.

The pressure is all in your head: A cilia-driven high-pressure pump in the head of a deep-sea animal

NASA Astrophysics Data System (ADS)

Nawroth, Janna; Katija, Kakani; Shelley, Michael; Kanso, Eva

2017-11-01

Motile cilia are microscopic, hair-like structures on the cell surface that can sense and propel the extracellular fluid environment. In many ciliated systems found in nature, such as the mammalian airways and marine sponges, the organization and collective behavior of the cilia favors the pumping of fluids at low pressures and high volumes. We recently discovered an alternate design located in the head of a deep-sea animal called Larvacean. Here, cilia morphology, kinematics and flow indicate a role in maintaining the hydrostatic skeleton of the animal by generating a high-pressure flow. We describe our empirical and computational approaches toward understanding the design principles and dynamic range of this newly discovered pumping mechanism. In ongoing work, we further explore the fluid dynamic constraints on the morphological diversity of cilia and the resulting categories of fluid transport functions.

What we can learn from a tadpole about ciliopathies and airway diseases: Using systems biology in Xenopus to study cilia and mucociliary epithelia.

PubMed

Walentek, Peter; Quigley, Ian K

2017-01-01

Over the past years, the Xenopus embryo has emerged as an incredibly useful model organism for studying the formation and function of cilia and ciliated epithelia in vivo. This has led to a variety of findings elucidating the molecular mechanisms of ciliated cell specification, basal body biogenesis, cilia assembly, and ciliary motility. These findings also revealed the deep functional conservation of signaling, transcriptional, post-transcriptional, and protein networks employed in the formation and function of vertebrate ciliated cells. Therefore, Xenopus research can contribute crucial insights not only into developmental and cell biology, but also into the molecular mechanisms underlying cilia related diseases (ciliopathies) as well as diseases affecting the ciliated epithelium of the respiratory tract in humans (e.g., chronic lung diseases). Additionally, systems biology approaches including transcriptomics, genomics, and proteomics have been rapidly adapted for use in Xenopus, and broaden the applications for current and future translational biomedical research. This review aims to present the advantages of using Xenopus for cilia research, highlight some of the evolutionarily conserved key concepts and mechanisms of ciliated cell biology that were elucidated using the Xenopus model, and describe the potential for Xenopus research to address unresolved questions regarding the molecular mechanisms of ciliopathies and airway diseases. © 2017 Wiley Periodicals, Inc.

Ccdc11 is a novel centriolar satellite protein essential for ciliogenesis and establishment of left-right asymmetry.

PubMed

Silva, Erica; Betleja, Ewelina; John, Emily; Spear, Philip; Moresco, James J; Zhang, Siwei; Yates, John R; Mitchell, Brian J; Mahjoub, Moe R

2016-01-01

The establishment of left-right (L-R) asymmetry in vertebrates is dependent on the sensory and motile functions of cilia during embryogenesis. Mutations in CCDC11 disrupt L-R asymmetry and cause congenital heart disease in humans, yet the molecular and cellular functions of the protein remain unknown. Here we demonstrate that Ccdc11 is a novel component of centriolar satellites-cytoplasmic granules that serve as recruitment sites for proteins destined for the centrosome and cilium. Ccdc11 interacts with core components of satellites, and its loss disrupts the subcellular organization of satellite proteins and perturbs primary cilium assembly. Ccdc11 colocalizes with satellite proteins in human multiciliated tracheal epithelia, and its loss inhibits motile ciliogenesis. Similarly, depletion of CCDC11 in Xenopus embryos causes defective assembly and motility of cilia in multiciliated epidermal cells. To determine the role of CCDC11 during vertebrate development, we generated mutant alleles in zebrafish. Loss of CCDC11 leads to defective ciliogenesis in the pronephros and within the Kupffer's vesicle and results in aberrant L-R axis determination. Our results highlight a critical role for Ccdc11 in the assembly and function of motile cilia and implicate centriolar satellite-associated proteins as a new class of proteins in the pathology of L-R patterning and congenital heart disease. © 2016 Silva, Betleja, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Genetic Analysis Reveals a Hierarchy of Interactions between Polycystin-Encoding Genes and Genes Controlling Cilia Function during Left-Right Determination

PubMed Central

Grimes, Daniel T.; Keynton, Jennifer L.; Buenavista, Maria T.; Jin, Xingjian; Patel, Saloni H.; Kyosuke, Shinohara; Williams, Debbie J.; Hamada, Hiroshi; Hussain, Rohanah; Nauli, Surya M.; Norris, Dominic P.

2016-01-01

During mammalian development, left-right (L-R) asymmetry is established by a cilia-driven leftward fluid flow within a midline embryonic cavity called the node. This ‘nodal flow’ is detected by peripherally-located crown cells that each assemble a primary cilium which contain the putative Ca2+ channel PKD2. The interaction of flow and crown cell cilia promotes left side-specific expression of Nodal in the lateral plate mesoderm (LPM). Whilst the PKD2-interacting protein PKD1L1 has also been implicated in L-R patterning, the underlying mechanism by which flow is detected and the genetic relationship between Polycystin function and asymmetric gene expression remains unknown. Here, we characterize a Pkd1l1 mutant line in which Nodal is activated bilaterally, suggesting that PKD1L1 is not required for LPM Nodal pathway activation per se, but rather to restrict Nodal to the left side downstream of nodal flow. Epistasis analysis shows that Pkd1l1 acts as an upstream genetic repressor of Pkd2. This study therefore provides a genetic pathway for the early stages of L-R determination. Moreover, using a system in which cultured cells are supplied artificial flow, we demonstrate that PKD1L1 is sufficient to mediate a Ca2+ signaling response after flow stimulation. Finally, we show that an extracellular PKD domain within PKD1L1 is crucial for PKD1L1 function; as such, destabilizing the domain causes L-R defects in the mouse. Our demonstration that PKD1L1 protein can mediate a response to flow coheres with a mechanosensation model of flow sensation in which the force of fluid flow drives asymmetric gene expression in the embryo. PMID:27272319

An update on the pathophysiology and management of polycystic liver disease.

PubMed

Wong, May Yw; McCaughan, Geoffrey W; Strasser, Simone I

2017-06-01

Polycystic liver disease (PLD) is characterized by the presence of multiple cholangiocyte-derived hepatic cysts that progressively replace liver tissue. They are classified as an inherited ciliopathy /cholangiopathy as pathology exists at the level of the primary cilia of cholangiocytes. Aberrant expression of the proteins in primary cilia can impair their structures and functions, thereby promoting cystogenesis. Areas covered: This review begins by looking at the epidemiology of PLD and its natural history. It then describes the pathophysiology and corresponding potential treatment strategies for PLD. Expert commentary: Traditionally, therapies for symptomatic PLD have been limited to symptomatic management and surgical interventions. Such techniques are not completely effective, do not alter the natural history of the disease, and are linked with high rate of re-accumulation of cysts. As a result, there has been a push for drugs targeted at abnormal cellular signaling cascades to address deregulated proliferation, cell dedifferentiation, apoptosis and fluid secretion. Currently, the only available drug treatments that halt disease progression and improve quality of life in PLD patients are somatostatin analogues. Numerous preclinical studies suggest that targeting components of the signaling pathways that influence cyst development can ameliorate growth of hepatic cysts.

Nephronophthisis: Disease Mechanisms of a Ciliopathy

PubMed Central

Hildebrandt, Friedhelm; Attanasio, Massimo; Otto, Edgar

2009-01-01

Nephronophthisis (NPHP), a recessive cystic kidney disease, is the most frequent genetic cause of end-stage kidney disease in children and young adults. Positional cloning of nine genes (NPHP1-9) and functional characterization of their encoded proteins (nephrocystins) has contributed to a unifying theory that defines cystic kidney diseases as “ciliopathies”. The theory is based on the finding that all proteins mutated in cystic kidney diseases of humans or animal models are expressed in primary cilia or centrosomes of renal epithelial cells. Primary cilia are sensory organelles that connect mechanosensory, visual, and other stimuli to mechanisms of epithelial cell polarity and cell cycle control. Mutations in NPHP genes cause defects in signaling mechanisms that involve the non-canonical Wnt signaling pathway and the sonic hedgehog signaling pathway, resulting in defects of planar cell polarity and tissue maintenance. The ciliary theory explains the multiple organ involvement in NPHP, which includes retinal degeneration, cerebellar hypoplasia, liver fibrosis, situs inversus, and mental retardation. Positional cloning of dozens of unknown genes that cause NPHP will elucidate further signaling mechanisms involved. Nephrocystins are highly conserved in evolution, thus allowing the use of animal models to develop future therapeutic approaches. PMID:19118152

Bardet-Biedl syndrome: Genetics, molecular pathophysiology, and disease management.

PubMed

Priya, Sathya; Nampoothiri, Sheela; Sen, Parveen; Sripriya, S

2016-09-01

Primary cilia play a key role in sensory perception and various signaling pathways. Any defect in them leads to group of disorders called ciliopathies, and Bardet-Biedl syndrome (BBS, OMIM 209900) is one among them. The disorder is clinically and genetically heterogeneous, with various primary and secondary clinical manifestations, and shows autosomal recessive inheritance and highly prevalent in inbred/consanguineous populations. The disease mapped to at least twenty different genes (BBS1-BBS20), follow oligogenic inheritance pattern. BBS proteins localizes to the centerosome and regulates the biogenesis and functions of the cilia. In BBS, the functioning of various systemic organs (with ciliated cells) gets deranged and results in systemic manifestations. Certain components of the disease (such as obesity, diabetes, and renal problems) when noticed earlier offer a disease management benefit to the patients. However, the awareness of the disease is comparatively low and most often noticed only after severe vision loss in patients, which is usually in the first decade of the patient's age. In the current review, we have provided the recent updates retrieved from various types of scientific literature through journals, on the genetics, its molecular relevance, and the clinical outcome in BBS. The review in nutshell would provide the basic awareness of the disease that will have an impact in disease management and counseling benefits to the patients and their families.

Flow Induced by Ex-Vivo Nasal Cilia: Developing an Index of Dyskinesis

NASA Astrophysics Data System (ADS)

Grotberg, James; Bottier, Mathieu; Pena-Fernandez, Marta; Blanchon, Sylvain; Pelle, Gabriel; Bequignon, Emilie; Isabey, Daniel; Coste, Andre; Escudier, Estelle; Papon, Jean-Francois; Filoche, Marcel; Louis, Bruno

2017-11-01

Mucociliary clearance is one of the major lines of defense of the respiratory system. The mucus layer coating the pulmonary airways is moved along and out of the lung by the activity of motile cilia, thus expelling the particles trapped in it. Here we compare ex vivomeasurements of a Newtonian flow induced by cilia beating (using micro-beads as tracers) and a mathematical model of this fluid flow. Samples of nasal epithelial cells placed in water are recorded by high-speed video-microscopy and ciliary beat pattern is inferred. Automatic tracking of micro-beads, used as markers of the flow generated by cilia motion, enables us also to assess the steady velocity profile as a function of the distance above the cilia. This profile is shown to be essentially parabolic. This compares well to a 2D mathematical model for ciliary fluid propulsion using an envelope model. From the model and the experimental measurements, the shear stress exerted by the cilia is deduced. Finally, this shear stress is proposed as a new index for characterizing the efficiency of ciliary beating and diagnosing dyskinesis.

Observing planar cell polarity in multiciliated mouse airway epithelial cells.

PubMed

Vladar, Eszter K; Lee, Yin Loon; Stearns, Tim; Axelrod, Jeffrey D

2015-01-01

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. Copyright © 2015 Elsevier Inc. All rights reserved.

The Gene Ontology of eukaryotic cilia and flagella.

PubMed

Roncaglia, Paola; van Dam, Teunis J P; Christie, Karen R; Nacheva, Lora; Toedt, Grischa; Huynen, Martijn A; Huntley, Rachael P; Gibson, Toby J; Lomax, Jane

2017-01-01

Recent research into ciliary structure and function provides important insights into inherited diseases termed ciliopathies and other cilia-related disorders. This wealth of knowledge needs to be translated into a computational representation to be fully exploitable by the research community. To this end, members of the Gene Ontology (GO) and SYSCILIA Consortia have worked together to improve representation of ciliary substructures and processes in GO. Members of the SYSCILIA and Gene Ontology Consortia suggested additions and changes to GO, to reflect new knowledge in the field. The project initially aimed to improve coverage of ciliary parts, and was then broadened to cilia-related biological processes. Discussions were documented in a public tracker. We engaged the broader cilia community via direct consultation and by referring to the literature. Ontology updates were implemented via ontology editing tools. So far, we have created or modified 127 GO terms representing parts and processes related to eukaryotic cilia/flagella or prokaryotic flagella. A growing number of biological pathways are known to involve cilia, and we continue to incorporate this knowledge in GO. The resulting expansion in GO allows more precise representation of experimentally derived knowledge, and SYSCILIA and GO biocurators have created 199 annotations to 50 human ciliary proteins. The revised ontology was also used to curate mouse proteins in a collaborative project. The revised GO and annotations, used in comparative 'before and after' analyses of representative ciliary datasets, improve enrichment results significantly. Our work has resulted in a broader and deeper coverage of ciliary composition and function. These improvements in ontology and protein annotation will benefit all users of GO enrichment analysis tools, as well as the ciliary research community, in areas ranging from microscopy image annotation to interpretation of high-throughput studies. We welcome feedback to further enhance the representation of cilia biology in GO.

Multiscale Modeling of Primary Cilium Deformations Under Local Forces and Shear Flows

NASA Astrophysics Data System (ADS)

Peng, Zhangli; Feng, Zhe; Resnick, Andrew; Young, Yuan-Nan

2017-11-01

We study the detailed deformations of a primary cilium under local forces and shear flows by developing a multiscale model based on the state-of-the-art understanding of its molecular structure. Most eukaryotic cells are ciliated with primary cilia. Primary cilia play important roles in chemosensation, thermosensation, and mechanosensation, but the detailed mechanism for mechanosensation is not well understood. We apply the dissipative particle dynamics (DPD) to model an entire well with a primary cilium and consider its different components, including the basal body, microtubule doublets, actin cortex, and lipid bilayer. We calibrate the mechanical properties of individual components and their interactions from experimental measurements and molecular dynamics simulations. We validate the simulations by comparing the deformation profile of the cilium and the rotation of the basal body with optical trapping experiments. After validations, we investigate the deformation of the primary cilium under shear flows. Furthermore, we calculate the membrane tensions and cytoskeleton stresses, and use them to predict the activation of mechanosensitive channels.

Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism

PubMed Central

Chen, Julia C.; Hoey, David A.; Chua, Mardonn; Bellon, Raymond; Jacobs, Christopher R.

2016-01-01

It has long been suspected, but never directly shown, that bone formed to accommodate an increase in mechanical loading is related to the creation of osteoblasts from skeletal stem cells. Indeed, biophysical stimuli potently regulate osteogenic lineage commitment in vitro. In this study, we transplanted bone marrow cells expressing green fluorescent protein, to enable lineage tracing, and subjected mice to a biophysical stimulus, to elicit a bone-forming response. We detected cells derived from transplanted progenitors embedded within the bone matrix near active bone-forming surfaces in response to loading, demonstrating for the first time, that mechanical signals enhance the homing and attachment of bone marrow cells to bone surfaces and the commitment to an osteogenic lineage of these cells in vivo. Furthermore, we used an inducible Cre/Lox recombination system to delete kinesin family member 3A (Kif3a), a gene that is essential for primary cilia formation, at will in transplanted cells and their progeny, regardless of which tissue may have incorporated them. Disruption of the mechanosensing organelle, the primary cilium in a progenitor population, significantly decreased the amount of bone formed in response to mechanical stimulation. The collective results of our study directly demonstrate that, in a novel experimental stem cell mechanobiology model, mechanical signals enhance osteogenic lineage commitment in vivo and that the primary cilium contributes to this process.—Chen, J. C., Hoey, D. A., Chua, M., Bellon, R., Jacobs, C. R. Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism. PMID:26675708

A novel murine allele of Intraflagellar Transport Protein 172 causes a syndrome including VACTERL-like features with hydrocephalus.

PubMed

Friedland-Little, Joshua M; Hoffmann, Andrew D; Ocbina, Polloneal Jymmiel R; Peterson, Mike A; Bosman, Joshua D; Chen, Yan; Cheng, Steven Y; Anderson, Kathryn V; Moskowitz, Ivan P

2011-10-01

The primary cilium is emerging as a crucial regulator of signaling pathways central to vertebrate development and human disease. We identified atrioventricular canal 1 (avc1), a mouse mutation that caused VACTERL association with hydrocephalus, or VACTERL-H. We showed that avc1 is a hypomorphic mutation of intraflagellar transport protein 172 (Ift172), required for ciliogenesis and Hedgehog (Hh) signaling. Phenotypically, avc1 caused VACTERL-H but not abnormalities in left-right (L-R) axis formation. Avc1 resulted in structural cilia defects, including truncated cilia in vivo and in vitro. We observed a dose-dependent requirement for Ift172 in ciliogenesis using an allelic series generated with Ift172(avc1) and Ift172(wim), an Ift172 null allele: cilia were present on 42% of avc1 mouse embryonic fibroblast (MEF) and 28% of avc1/wim MEFs, in contrast to >90% of wild-type MEFs. Furthermore, quantitative cilium length analysis identified two specific cilium populations in mutant MEFS: a normal population with normal IFT and a truncated population, 50% of normal length, with disrupted IFT. Cells from wild-type embryos had predominantly full-length cilia, avc1 embryos, with Hh signaling abnormalities but not L-R abnormalities, had cilia equally divided between full-length and truncated, and avc1/wim embryos, with both Hh signaling and L-R abnormalities, were primarily truncated. Truncated Ift172 mutant cilia showed defects of the distal ciliary axoneme, including disrupted IFT88 localization and Hh-dependent Gli2 localization. We propose a model in which mutation of Ift172 results in a specific class of abnormal cilia, causing disrupted Hh signaling while maintaining L-R axis determination, and resulting in the VACTERL-H phenotype.

A novel mutation causing nephronophthisis in the Lewis polycystic kidney rat localises to a conserved RCC1 domain in Nek8

PubMed Central

2012-01-01

Background Nephronophthisis (NPHP) as a cause of cystic kidney disease is the most common genetic cause of progressive renal failure in children and young adults. NPHP is characterized by abnormal and/or loss of function of proteins associated with primary cilia. Previously, we characterized an autosomal recessive phenotype of cystic kidney disease in the Lewis Polycystic Kidney (LPK) rat. Results In this study, quantitative trait locus analysis was used to define a ~1.6Mbp region on rat chromosome 10q25 harbouring the lpk mutation. Targeted genome capture and next-generation sequencing of this region identified a non-synonymous mutation R650C in the NIMA (never in mitosis gene a)- related kinase 8 ( Nek8) gene. This is a novel Nek8 mutation that occurs within the regulator of chromosome condensation 1 (RCC1)-like region of the protein. Specifically, the R650C substitution is located within a G[QRC]LG repeat motif of the predicted seven bladed beta-propeller structure of the RCC1 domain. The rat Nek8 gene is located in a region syntenic to portions of human chromosome 17 and mouse 11. Scanning electron microscopy confirmed abnormally long cilia on LPK kidney epithelial cells, and fluorescence immunohistochemistry for Nek8 protein revealed altered cilia localisation. Conclusions When assessed relative to other Nek8 NPHP mutations, our results indicate the whole propeller structure of the RCC1 domain is important, as the different mutations cause comparable phenotypes. This study establishes the LPK rat as a novel model system for NPHP and further consolidates the link between cystic kidney disease and cilia proteins. PMID:22899815

The Oak Ridge Polycystic Kidney mouse: modeling ciliopathies of mice and men.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lehman, J M; Michaud III, Edward J; Schoeb, T

2008-08-01

The Oak Ridge Polycystic Kidney (ORPK) mouse was described nearly 14 years ago as a model for human recessive polycystic kidney disease. The ORPK mouse arose through integration of a transgene into an intron of the Ift88 gene resulting in a hypomorphic allele (Ift88Tg737Rpw). The Ift88Tg737Rpw mutation impairs intraflagellar transport (IFT), a process required for assembly of motile and immotile cilia. Historically, the primary immotile cilium was thought to have minimal importance for human health; however, a rapidly expanding number of human disorders have now been attributed to ciliary defects. Importantly, many of these phenotypes are present and can bemore » analyzed using the ORPK mouse. In this review, we highlight the research conducted using the OPRK mouse and the phenotypes shared with human cilia disorders. Furthermore, we describe an additional follicular dysplasia phenotype in the ORPK mouse, which alongside the ectodermal dysplasias seen in human Ellis-van Creveld and Sensenbrenner's syndromes, suggests an unappreciated role for primary cilia in the skin and hair follicle.« less

Intraflagellar transport protein 122 antagonizes Sonic Hedgehog signaling and controls ciliary localization of pathway components.

PubMed

Qin, Jian; Lin, Yulian; Norman, Ryan X; Ko, Hyuk W; Eggenschwiler, Jonathan T

2011-01-25

Primary cilia are required for proper Sonic Hedgehog (Shh) signaling in mammals. However, their role in the signal transduction process remains unclear. We have identified sister of open brain (sopb), a null allele of mouse Intraflagellar transport protein 122 (Ift122). IFT122 negatively regulates the Shh pathway in the cilium at a step downstream of the Shh ligand and the transmembrane protein Smoothened, but upstream of the Gli2 transcription factor. Ift122(sopb) mutants generate primary cilia, but they show features of defective retrograde intraflagellar transport. IFT122 controls the ciliary localization of Shh pathway regulators in different ways. Disruption of IFT122 leads to accumulation of Gli2 and Gli3 at cilia tips while blocking the ciliary localization of the antagonist TULP3. Suppressor of Fused and Smoothened localize to the cilium through an IFT122-independent mechanism. We propose that the balance between positive and negative regulators of the Shh pathway at the cilium tip controls the output of the pathway and that Shh signaling regulates this balance through intraflagellar transport.

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3

PubMed Central

Olcese, Chiara; Patel, Mitali P.; Shoemark, Amelia; Kiviluoto, Santeri; Legendre, Marie; Williams, Hywel J.; Vaughan, Cara K.; Hayward, Jane; Goldenberg, Alice; Emes, Richard D.; Munye, Mustafa M.; Dyer, Laura; Cahill, Thomas; Bevillard, Jeremy; Gehrig, Corinne; Guipponi, Michel; Chantot, Sandra; Duquesnoy, Philippe; Thomas, Lucie; Jeanson, Ludovic; Copin, Bruno; Tamalet, Aline; Thauvin-Robinet, Christel; Papon, Jean- François; Garin, Antoine; Pin, Isabelle; Vera, Gabriella; Aurora, Paul; Fassad, Mahmoud R.; Jenkins, Lucy; Boustred, Christopher; Cullup, Thomas; Dixon, Mellisa; Onoufriadis, Alexandros; Bush, Andrew; Chung, Eddie M. K.; Antonarakis, Stylianos E.; Loebinger, Michael R.; Wilson, Robert; Armengot, Miguel; Escudier, Estelle; Hogg, Claire; Al-Turki, Saeed; Anderson, Carl; Antony, Dinu; Barroso, Inês; Beales, Philip L.; Bentham, Jamie; Bhattacharya, Shoumo; Carss, Keren; Chatterjee, Krishna; Cirak, Sebahattin; Cosgrove, Catherine; Allan, Daly; Durbin, Richard; Fitzpatrick, David; Floyd, Jamie; Foley, A. Reghan; Franklin, Chris; Futema, Marta; Humphries, Steve E.; Hurles, Matt; McCarthy, Shane; Muddyman, Dawn; Muntoni, Francesco; Parker, Victoria; Payne, Felicity; Plagnol, Vincent; Raymond, Lucy; Savage, David B.; Scambler, Peter J.; Schmidts, Miriam; Semple, Robert; Serra, Eva; Stalker, Jim; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Walter, Klaudia; Amselem, Serge; Sun, Zhaoxia; Bartoloni, Lucia; Blouin, Jean-Louis; Mitchison, Hannah M.

2017-01-01

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins. PMID:28176794

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3.

PubMed

Olcese, Chiara; Patel, Mitali P; Shoemark, Amelia; Kiviluoto, Santeri; Legendre, Marie; Williams, Hywel J; Vaughan, Cara K; Hayward, Jane; Goldenberg, Alice; Emes, Richard D; Munye, Mustafa M; Dyer, Laura; Cahill, Thomas; Bevillard, Jeremy; Gehrig, Corinne; Guipponi, Michel; Chantot, Sandra; Duquesnoy, Philippe; Thomas, Lucie; Jeanson, Ludovic; Copin, Bruno; Tamalet, Aline; Thauvin-Robinet, Christel; Papon, Jean-François; Garin, Antoine; Pin, Isabelle; Vera, Gabriella; Aurora, Paul; Fassad, Mahmoud R; Jenkins, Lucy; Boustred, Christopher; Cullup, Thomas; Dixon, Mellisa; Onoufriadis, Alexandros; Bush, Andrew; Chung, Eddie M K; Antonarakis, Stylianos E; Loebinger, Michael R; Wilson, Robert; Armengot, Miguel; Escudier, Estelle; Hogg, Claire; Amselem, Serge; Sun, Zhaoxia; Bartoloni, Lucia; Blouin, Jean-Louis; Mitchison, Hannah M

2017-02-08

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2-DNAAF4-HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.

Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways.

PubMed

Geneva, Ivayla I; Tan, Han Yen; Calvert, Peter D

2017-02-15

Resolution limitations of optical systems are major obstacles for determining whether proteins are enriched within cell compartments. Here we use an approach to determine the degree of membrane protein ciliary enrichment that quantitatively accounts for the differences in sampling of the ciliary and apical membranes inherent to confocal microscopes. Theory shows that cilia will appear more than threefold brighter than the surrounding apical membrane when the densities of fluorescently labeled proteins are the same, thus providing a benchmark for ciliary enrichment. Using this benchmark, we examined the ciliary enrichment signals of two G protein-coupled receptors (GPCRs)-the somatostatin receptor 3 and rhodopsin. Remarkably, we found that the C-terminal VxPx motif, required for efficient enrichment of rhodopsin within rod photoreceptor sensory cilia, inhibited enrichment of the somatostatin receptor in primary cilia. Similarly, VxPx inhibited primary cilium enrichment of a chimera of rhodopsin and somatostatin receptor 3, where the dual Ax(S/A)xQ ciliary targeting motifs within the third intracellular loop of the somatostatin receptor replaced the third intracellular loop of rhodopsin. Rhodopsin was depleted from primary cilia but gained access, without being enriched, with the dual Ax(S/A)xQ motifs. Ciliary enrichment of these GPCRs thus operates via distinct mechanisms in different cells. © 2017 Geneva et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Multiciliated Cells in Animals.

PubMed

Meunier, Alice; Azimzadeh, Juliette

2016-12-01

Many animal cells assemble single cilia involved in motile and/or sensory functions. In contrast, multiciliated cells (MCCs) assemble up to 300 motile cilia that beat in a coordinate fashion to generate a directional fluid flow. In the human airways, the brain, and the oviduct, MCCs allow mucus clearance, cerebrospinal fluid circulation, and egg transportation, respectively. Impairment of MCC function leads to chronic respiratory infections and increased risks of hydrocephalus and female infertility. MCC differentiation during development or repair involves the activation of a regulatory cascade triggered by the inhibition of Notch activity in MCC progenitors. The downstream events include the simultaneous assembly of a large number of basal bodies (BBs)-from which cilia are nucleated-in the cytoplasm of the differentiating MCCs, their migration and docking at the plasma membrane associated to an important remodeling of the actin cytoskeleton, and the assembly and polarization of motile cilia. The direction of ciliary beating is coordinated both within cells and at the tissue level by a combination of planar polarity cues affecting BB position and hydrodynamic forces that are both generated and sensed by the cilia. Herein, we review the mechanisms controlling the specification and differentiation of MCCs and BB assembly and organization at the apical surface, as well as ciliary assembly and coordination in MCCs. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

Zebrafish mutations affecting cilia motility share similar cystic phenotypes and suggest a mechanism of cyst formation that differs from pkd2 morphants

PubMed Central

Sullivan-Brown, Jessica; Schottenfeld, Jodi; Okabe, Noriko; Hostetter, Christine L.; Serluca, Fabrizio C.; Thiberge, Stephan Y.; Burdine, Rebecca D.

2008-01-01

Zebrafish are an attractive model for studying the earliest cellular defects occurring during renal cyst formation because its kidney (the pronephros) is simple and genes that cause cystic kidney diseases (CKD) in humans, cause pronephric dilations in zebrafish. By comparing phenotypes in three different mutants, locke, swt and kurly, we find that dilations occur prior to 48 hpf in the medial tubules, a location similar to where cysts form in some mammalian diseases. We demonstrate that the first observeable phenotypes associated with dilation include cilia motility and luminal remodeling defects. Importantly, we show that some phenotypes common to human CKD, such as an increased number of cells, are secondary consequences of dilation. Despite having differences in cilia motility, locke, swt and kurly share similar cystic phenotypes, suggesting that they function in a common pathway. To begin to understand the molecular mechanisms involved in cyst formation, we have cloned the swt mutation and find that it encodes a novel leucine rich repeat containing protein (LRRC50), which is thought to function in correct dynein assembly in cilia. Finally, we show that knockdown of polycystic kidney disease 2 (pkd2) specifically causes glomerular cysts and does not affect cilia motility, suggesting multiple mechanisms exist for cyst formation. PMID:18178183

Neuronal NOS localises to human airway cilia.

PubMed

Jackson, Claire L; Lucas, Jane S; Walker, Woolf T; Owen, Holly; Premadeva, Irnthu; Lackie, Peter M

2015-01-30

Airway NO synthase (NOS) isoenzymes are responsible for rapid and localised nitric oxide (NO) production and are expressed in airway epithelium. We sought to determine the localisation of neuronal NOS (nNOS) in airway epithelium due to the paucity of evidence. Sections of healthy human bronchial tissue in glycol methacrylate resin and human nasal polyps in paraffin wax were immunohistochemically labelled and reproducibly demonstrated nNOS immunoreactivity, particularly at the proximal portion of cilia; this immunoreactivity was blocked by a specific nNOS peptide fragment. Healthy human epithelial cells differentiated at an air-liquid interface (ALI) confirmed the presence of all three NOS isoenzymes by immunofluorescence labelling. Only nNOS immunoreactivity was specific to the ciliary axonemeand co-localised with the cilia marker β-tubulin in the proximal part of the ciliary axoneme. We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia function. Copyright © 2014 Elsevier Inc. All rights reserved.

IFT46 plays an essential role in cilia development

PubMed Central

Lee, Mi-Sun; Hwang, Kyu-Seok; Oh, Hyun-Woo; Ji-Ae, Kim; Kim, Hyun-Taek; Cho, Hyun-Soo; Lee, Jeong-Ju; Ko, Je Yeong; Choi, Jung-Hwa; Jeong, Yun-Mi; You, Kwan-Hee; Kim, Joon; Park, Doo-Sang; Nam, Ki-Hoan; Aizawa, Shinichi; Kiyonari, Hiroshi; Shioi, Go; Park, Jong-Hoon; Zhou, Weibin; Kim, Nam-Soon; Kim, Cheol-Hee

2015-01-01

Cilia are microtubule-based structures that project into the extracellular space. Ciliary defects are associated with several human diseases, including polycystic kidney disease, primary ciliary dyskinesia, left-right axis patterning, hydrocephalus and retinal degeneration. However, the genetic and cellular biological control of ciliogenesis remains poorly understood. The IFT46 is one of the highly conserved intraflagellar transport complex B proteins. In zebrafish, ift46 is expressed in various ciliated tissues such as Kupffer’s vesicle, pronephric ducts, ears and spinal cord. We show that ift46 is localized to the basal body. Knockdown of ift46 gene results in multiple phenotypes associated with various ciliopathies including kidney cysts, pericardial edema and ventral axis curvature. In ift46 morphants, cilia in kidney and spinal canal are shortened and abnormal. Similar ciliary defects are observed in otic vesicles, lateral line hair cells, olfactory pits, but not in Kupffer’s vesicle. To explore the functions of Ift46 during mouse development, we have generated Ift46 knock-out mice. The Ift46 mutants have developmental defects in brain, neural tube and heart. In particular Ift46(−/−) homozygotes displays randomization of the embryo heart looping, which is a hallmark of defective left-right (L/R) axis patterning. Taken together, our results demonstrated that IFT46 has an essential role in vertebrate ciliary development. PMID:25722189

Ciliopathies: The Trafficking Connection

PubMed Central

Madhivanan, Kayalvizhi; Aguilar, R. Claudio

2014-01-01

The primary cilium (PC) is a very dynamic hair-like membrane structure that assembles/disassembles in a cell-cycle dependent manner and is present in almost every cell type. Despite being continuous with the plasma membrane, a diffusion barrier located at the ciliary base confers the PC properties of a separate organelle with very specific characteristics and membrane composition. Therefore, vesicle trafficking is the major process by which components are acquired for cilium formation and maintenance. In fact, a system of specific sorting signals controls the right of cargo admission into the cilia. Disruption to the ciliary structure or its function leads to multi-organ diseases known as ciliopathies. These illnesses arise from a spectrum of mutations in any of the more than 50 loci linked to these conditions. Therefore, it is not surprising that symptom variability (specific manifestations and severity) among and within ciliopathies seems to be an emerging characteristic. Nevertheless, one can speculate that mutations occurring in genes whose products contribute to the overall vesicle trafficking to the PC (i.e., affecting cilia assembly) will lead to more severe symptoms, while those involved in the transport of specific cargoes will result in milder phenotypes. In this review, we summarize the trafficking mechanisms to the cilia and also provide a description of the trafficking defects observed in some ciliopathies which can be correlated to the severity of the pathology. PMID:25040720

Centrosomal protein Dzip1l binds Cby, promotes ciliary bud formation, and acts redundantly with Bromi to regulate ciliogenesis in the mouse.

PubMed

Wang, Chengbing; Li, Jia; Takemaru, Ken-Ichi; Jiang, Xiaogang; Xu, Guoqiang; Wang, Baolin

2018-03-15

The primary cilium is a microtubule-based organelle required for Hedgehog (Hh) signaling and consists of a basal body, a ciliary axoneme and a compartment between the first two structures, called the transition zone (TZ). The TZ serves as a gatekeeper to control protein composition in cilia, but less is known about its role in ciliary bud formation. Here, we show that centrosomal protein Dzip1l is required for Hh signaling between Smoothened and Sufu. Dzip1l colocalizes with basal body appendage proteins and Rpgrip1l, a TZ protein. Loss of Dzip1l results in reduced ciliogenesis and dysmorphic cilia in vivo Dzip1l interacts with, and acts upstream of, Cby, an appendage protein, in ciliogenesis. Dzip1l also has overlapping functions with Bromi (Tbc1d32) in ciliogenesis, cilia morphogenesis and neural tube patterning. Loss of Dzip1l arrests ciliogenesis at the stage of ciliary bud formation from the TZ. Consistent with this, Dzip1l mutant cells fail to remove the capping protein Cp110 (Ccp110) from the distal end of mother centrioles and to recruit Rpgrip1l to the TZ. Therefore, Dzip1l promotes ciliary bud formation and is required for the integrity of the TZ. © 2018. Published by The Company of Biologists Ltd.

Kinesin 1 regulates cilia length through an interaction with the Bardet-Biedl syndrome related protein CCDC28B.

PubMed

Novas, Rossina; Cardenas-Rodriguez, Magdalena; Lepanto, Paola; Fabregat, Matías; Rodao, Magela; Fariello, María Inés; Ramos, Mauricio; Davison, Camila; Casanova, Gabriela; Alfaya, Lucía; Lecumberry, Federico; González-Sapienza, Gualberto; Irigoín, Florencia; Badano, Jose L

2018-02-14

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, polydactyly, renal disease and mental retardation. CCDC28B is a BBS-associated protein that we have previously shown plays a role in cilia length regulation whereby its depletion results in shortened cilia both in cells and Danio rerio (zebrafish). At least part of that role is achieved by its interaction with the mTORC2 component SIN1, but the mechanistic details of this interaction and/or additional functions that CCDC28B might play in the context of cilia remain poorly understood. Here we uncover a novel interaction between CCDC28B and the kinesin 1 molecular motor that is relevant to cilia. CCDC28B interacts with kinesin light chain 1 (KLC1) and the heavy chain KIF5B. Notably, depletion of these kinesin 1 components results in abnormally elongated cilia. Furthermore, through genetic interaction studies we demonstrate that kinesin 1 regulates ciliogenesis through CCDC28B. We show that kinesin 1 regulates the subcellular distribution of CCDC28B, unexpectedly, inhibiting its nuclear accumulation, and a ccdc28b mutant missing a nuclear localization motif fails to rescue the phenotype in zebrafish morphant embryos. Therefore, we uncover a previously unknown role of kinesin 1 in cilia length regulation that relies on the BBS related protein CCDC28B.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays.

PubMed

Windpassinger, Christian; Piard, Juliette; Bonnard, Carine; Alfadhel, Majid; Lim, Shuhui; Bisteau, Xavier; Blouin, Stéphane; Ali, Nur'Ain B; Ng, Alvin Yu Jin; Lu, Hao; Tohari, Sumanty; Talib, S Zakiah A; van Hul, Noémi; Caldez, Matias J; Van Maldergem, Lionel; Yigit, Gökhan; Kayserili, Hülya; Youssef, Sameh A; Coppola, Vincenzo; de Bruin, Alain; Tessarollo, Lino; Choi, Hyungwon; Rupp, Verena; Roetzer, Katharina; Roschger, Paul; Klaushofer, Klaus; Altmüller, Janine; Roy, Sudipto; Venkatesh, Byrappa; Ganger, Rudolf; Grill, Franz; Ben Chehida, Farid; Wollnik, Bernd; Altunoglu, Umut; Al Kaissi, Ali; Reversade, Bruno; Kaldis, Philipp

2017-09-07

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development. Copyright © 2017 American Society of Human Genetics. All rights reserved.

C2cd3 is required for cilia formation and Hedgehog signaling in mouse

PubMed Central

Hoover, Amber N.; Wynkoop, Aaron; Zeng, Huiqing; Jia, Jinping; Niswander, Lee A.; Liu, Aimin

2011-01-01

Cilia are essential for mammalian embryonic development as well as for the physiological activity of various adult organ systems. Despite the multiple crucial roles that cilia play, the mechanisms underlying ciliogenesis in mammals remain poorly understood. Taking a forward genetic approach, we have identified Hearty (Hty), a recessive lethal mouse mutant with multiple defects, including neural tube defects, abnormal dorsal-ventral patterning of the spinal cord, a defect in left-right axis determination and severe polydactyly (extra digits). By genetic mapping, sequence analysis of candidate genes and characterization of a second mutant allele, we identify Hty as C2cd3, a novel gene encoding a vertebrate-specific C2 domain-containing protein. Target gene expression and double-mutant analyses suggest that C2cd3 is an essential regulator of intracellular transduction of the Hedgehog signal. Furthering a link between Hedgehog signaling and cilia function, we find that cilia formation and proteolytic processing of Gli3 are disrupted in C2cd3 mutants. Finally, we observe C2cd3 protein at the basal body, consistent with its essential function in ciliogenesis. Interestingly, the human ortholog for this gene lies in proximity to the critical regions of Meckel-Gruber syndrome 2 (MKS2) and Joubert syndrome 2 (JBTS2), making it a potential candidate for these two human genetic disorders. PMID:19004860

Handedness and situs inversus in primary ciliary dyskinesia.

PubMed Central

McManus, I. C.; Martin, N.; Stubbings, G. F.; Chung, E. M. K.; Mitchison, H. M.

2004-01-01

...The limbs on the right side are stronger. [The] cause may be ... [that] ... motion, and abilities of moving, are somewhat holpen from the liver, which lieth on the right side. (Sir Francis Bacon, Sylva sylvarum (1627).)Fifty per cent of people with primary ciliary dyskinesia (PCD) (also known as immotile cilia syndrome or Siewert-Kartagener syndrome) have situs inversus, which is thought to result from absent nodal ciliary rotation and failure of normal symmetry breaking. In a study of 88 people with PCD, only 15.2% of 46 individuals with situs inversus, and 14.3% of 42 individuals with situs solitus, were left handed. Because cerebral lateralization is therefore still present, the nodal cilia cannot be the primary mechanism responsible for symmetry breaking in the vertebrate body. Intriguingly, one behavioural lateralization, wearing a wrist-watch on the right wrist, did correlate with situs inversus. PMID:15615683

Handedness and situs inversus in primary ciliary dyskinesia.

PubMed

McManus, I C; Martin, N; Stubbings, G F; Chung, E M K; Mitchison, H M

2004-12-22

... The limbs on the right side are stronger. [The] cause may be ... [that] ... motion, and abilities of moving, are somewhat holpen from the liver, which lieth on the right side. (Sir Francis Bacon, Sylva sylvarum (1627).)Fifty per cent of people with primary ciliary dyskinesia (PCD) (also known as immotile cilia syndrome or Siewert-Kartagener syndrome) have situs inversus, which is thought to result from absent nodal ciliary rotation and failure of normal symmetry breaking. In a study of 88 people with PCD, only 15.2% of 46 individuals with situs inversus, and 14.3% of 42 individuals with situs solitus, were left handed. Because cerebral lateralization is therefore still present, the nodal cilia cannot be the primary mechanism responsible for symmetry breaking in the vertebrate body. Intriguingly, one behavioural lateralization, wearing a wrist-watch on the right wrist, did correlate with situs inversus.

Calcium signals act through histone deacetylase to mediate pronephric kidney morphogenesis.

PubMed

Rothschild, Sarah C; Lee, Hunter J; Ingram, Sarah R; Mohammadi, Daniel K; Walsh, Gregory S; Tombes, Robert M

2018-06-01

Autosomal dominant polycystic kidney disease is the most common monogenetic kidney disorder and is linked to mutations in PKD1 and PKD2. PKD2, a Ca 2+ -conducting TRP channel enriched in ciliated cells and gated by extracellular signals, is necessary to activate the multifunctional Ca 2+/ calmodulin-dependent protein kinase type 2 (CaMK-II), enabling kidney morphogenesis and cilia stability. In this study, antisense morpholino oligonucleotides and pharmacological compounds were employed to investigate the roles of class II HDAC family members (HDAC 4, 5, and 6) in Zebrafish kidney development. While all three class II HDAC genes were expressed throughout the embryo during early development, HDAC5-morphant embryos exhibited anterior cysts and destabilized cloacal cilia, similar to PKD2 and CaMK-II morphants. In contrast, HDAC4-morphant embryos exhibited elongated cloacal cilia and lacked anterior kidney defects. Suppression of HDAC4 partially reversed the cilia shortening and anterior convolution defects caused by CaMK-II deficiency, whereas HDAC5 loss exacerbated these defects. EGFP-HDAC4, but not EGFP-HDAC5, translocated into the nucleus upon CaMK-II suppression in pronephric kidney cells. These results support a model by which activated CaMK-II sequesters HDAC4 in the cytosol to enable primary cilia formation and kidney morphogenesis. Developmental Dynamics 247:807-817, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Mother Centriole Distal Appendages Mediate Centrosome Docking at the Immunological Synapse and Reveal Mechanistic Parallels with Ciliogenesis.

PubMed

Stinchcombe, Jane C; Randzavola, Lyra O; Angus, Karen L; Mantell, Judith M; Verkade, Paul; Griffiths, Gillian M

2015-12-21

Cytotoxic T lymphocytes (CTLs) are highly effective serial killers capable of destroying virally infected and cancerous targets by polarized release from secretory lysosomes. Upon target contact, the CTL centrosome rapidly moves to the immunological synapse, focusing microtubule-directed release at this point [1-3]. Striking similarities have been noted between centrosome polarization at the synapse and basal body docking during ciliogenesis [1, 4-8], suggesting that CTL centrosomes might dock with the plasma membrane during killing, in a manner analogous to primary cilia formation [1, 4]. However, questions remain regarding the extent and function of centrosome polarization at the synapse, and recent reports have challenged its role [9, 10]. Here, we use high-resolution transmission electron microscopy (TEM) tomography analysis to show that, as in ciliogenesis, the distal appendages of the CTL mother centriole contact the plasma membrane directly during synapse formation. This is functionally important as small interfering RNA (siRNA) targeting of the distal appendage protein, Cep83, required for membrane contact during ciliogenesis [11], impairs CTL secretion. Furthermore, the regulatory proteins CP110 and Cep97, which must dissociate from the mother centriole to allow cilia formation [12], remain associated with the mother centriole in CTLs, and neither axoneme nor transition zone ciliary structures form. Moreover, complete centrosome docking can occur in proliferating CTLs with multiple centriole pairs. Thus, in CTLs, centrosomes dock transiently with the membrane, within the cell cycle and without progression into ciliogenesis. We propose that this transient centrosome docking without cilia formation is important for CTLs to deliver rapid, repeated polarized secretion directed by the centrosome. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Sentan: A Novel Specific Component of the Apical Structure of Vertebrate Motile Cilia

PubMed Central

Yuba-Kubo, Akiko; Tsukita, Sachiko; Tsukita, Shoichiro; Amagai, Masayuki

2008-01-01

Human respiratory and oviductal cilia have specific apical structures characterized by a narrowed distal portion and a ciliary crown. These structures are conserved among vertebrates that have air respiration systems; however, the molecular components of these structures have not been defined, and their functions are unknown. To identify the molecular component(s) of the cilia apical structure, we screened EST libraries to identify gene(s) that are exclusively expressed in ciliated tissues, are transcriptionally up-regulated during in vitro ciliogenesis, and are not expressed in testis (because sperm flagella have no such apical structures). One of the identified gene products, named sentan, was localized to the distal tip region of motile cilia. Using anti-sentan polyclonal antibodies and electron microscopy, sentan was shown to localize exclusively to the bridging structure between the cell membrane and peripheral singlet microtubules, which specifically exists in the narrowed distal portion of cilia. Exogenously expressed sentan showed affinity for the membrane protrusions, and a protein–lipid binding assay revealed that sentan bound to phosphatidylserine. These findings suggest that sentan is the first molecular component of the ciliary tip to bridge the cell membrane and peripheral singlet microtubules, making the distal portion of the cilia narrow and stiff to allow for better airway clearance or ovum transport. PMID:18829862

A new index for characterizing micro-bead motion in a flow induced by ciliary beating: Part I, experimental analysis.

PubMed

Bottier, Mathieu; Blanchon, Sylvain; Pelle, Gabriel; Bequignon, Emilie; Isabey, Daniel; Coste, André; Escudier, Estelle; Grotberg, James B; Papon, Jean-François; Filoche, Marcel; Louis, Bruno

2017-07-01

Mucociliary clearance is one of the major lines of defense of the respiratory system. The mucus layer coating the pulmonary airways is moved along and out of the lung by the activity of motile cilia, thus expelling the particles trapped in it. Here we compare ex vivo measurements of a Newtonian flow induced by cilia beating (using micro-beads as tracers) and a mathematical model of this fluid flow, presented in greater detail in a second companion article. Samples of nasal epithelial cells placed in water are recorded by high-speed video-microscopy and ciliary beat pattern is inferred. Automatic tracking of micro-beads, used as markers of the flow generated by cilia motion, enables us also to assess the velocity profile as a function of the distance above the cilia. This profile is shown to be essentially parabolic. The obtained experimental data are used to feed a 2D mathematical and numerical model of the coupling between cilia, fluid, and micro-bead motion. From the model and the experimental measurements, the shear stress exerted by the cilia is deduced. Finally, this shear stress, which can easily be measured in the clinical setting, is proposed as a new index for characterizing the efficiency of ciliary beating.

A new index for characterizing micro-bead motion in a flow induced by ciliary beating: Part I, experimental analysis

PubMed Central

Bottier, Mathieu; Blanchon, Sylvain; Pelle, Gabriel; Bequignon, Emilie; Coste, André; Escudier, Estelle; Grotberg, James B.; Papon, Jean-François

2017-01-01

Mucociliary clearance is one of the major lines of defense of the respiratory system. The mucus layer coating the pulmonary airways is moved along and out of the lung by the activity of motile cilia, thus expelling the particles trapped in it. Here we compare ex vivo measurements of a Newtonian flow induced by cilia beating (using micro-beads as tracers) and a mathematical model of this fluid flow, presented in greater detail in a second companion article. Samples of nasal epithelial cells placed in water are recorded by high-speed video-microscopy and ciliary beat pattern is inferred. Automatic tracking of micro-beads, used as markers of the flow generated by cilia motion, enables us also to assess the velocity profile as a function of the distance above the cilia. This profile is shown to be essentially parabolic. The obtained experimental data are used to feed a 2D mathematical and numerical model of the coupling between cilia, fluid, and micro-bead motion. From the model and the experimental measurements, the shear stress exerted by the cilia is deduced. Finally, this shear stress, which can easily be measured in the clinical setting, is proposed as a new index for characterizing the efficiency of ciliary beating. PMID:28708889

Robust and stretchable indium gallium zinc oxide-based electronic textiles formed by cilia-assisted transfer printing

PubMed Central

Yoon, Jongwon; Jeong, Yunkyung; Kim, Heeje; Yoo, Seonggwang; Jung, Hoon Sun; Kim, Yonghun; Hwang, Youngkyu; Hyun, Yujun; Hong, Woong-Ki; Lee, Byoung Hun; Choa, Sung-Hoon; Ko, Heung Cho

2016-01-01

Electronic textile (e-textile) allows for high-end wearable electronic devices that provide easy access for carrying, handling and using. However, the related technology does not seem to be mature because the woven fabric hampers not only the device fabrication process directly on the complex surface but also the transfer printing of ultrathin planar electronic devices. Here we report an indirect method that enables conformal wrapping of surface with arbitrary yet complex shapes. Artificial cilia are introduced in the periphery of electronic devices as adhesive elements. The cilia also play an important role in confining a small amount of glue and damping mechanical stress to maintain robust electronic performance under mechanical deformation. The example of electronic applications depicts the feasibility of cilia for ‘stick-&-play' systems, which provide electronic functions by transfer printing on unconventional complex surfaces. PMID:27248982

Automatic analysis of ciliary beat frequency using optical flow

NASA Astrophysics Data System (ADS)

Figl, Michael; Lechner, Manuel; Werther, Tobias; Horak, Fritz; Hummel, Johann; Birkfellner, Wolfgang

2012-02-01

Ciliary beat frequency (CBF) can be a useful parameter for diagnosis of several diseases, as e.g. primary ciliary dyskinesia. (PCD). CBF computation is usually done using manual evaluation of high speed video sequences, a tedious, observer dependent, and not very accurate procedure. We used the OpenCV's pyramidal implementation of the Lukas-Kanade algorithm for optical flow computation and applied this to certain objects to follow the movements. The objects were chosen by their contrast applying the corner detection by Shi and Tomasi. Discrimination between background/noise and cilia by a frequency histogram allowed to compute the CBF. Frequency analysis was done using the Fourier transform in matlab. The correct number of Fourier summands was found by the slope in an approximation curve. The method showed to be usable to distinguish between healthy and diseased samples. However there remain difficulties in automatically identifying the cilia, and also in finding enough high contrast cilia in the image. Furthermore the some of the higher contrast cilia are lost (and sometimes found) by the method, an easy way to distinguish the correct sub-path of a point's path have yet to be found in the case where the slope methods doesn't work.

Regulation of Cilium Length and Intraflagellar Transport by the RCK-Kinases ICK and MOK in Renal Epithelial Cells

PubMed Central

Broekhuis, Joost R.; Verhey, Kristen J.; Jansen, Gert

2014-01-01

Primary cilia are important sensory organelles. They exist in a wide variety of lengths, which could reflect different cell-specific functions. How cilium length is regulated is unclear, but it probably involves intraflagellar transport (IFT), which transports protein complexes along the ciliary axoneme. Studies in various organisms have identified the small, conserved family of ros-cross hybridizing kinases (RCK) as regulators of cilium length. Here we show that Intestinal Cell Kinase (ICK) and MAPK/MAK/MRK overlapping kinase (MOK), two members of this family, localize to cilia of mouse renal epithelial (IMCD-3) cells and negatively regulate cilium length. To analyze the effects of ICK and MOK on the IFT machinery, we set up live imaging of five fluorescently tagged IFT proteins: KIF3B, a subunit of kinesin-II, the main anterograde IFT motor, complex A protein IFT43, complex B protein IFT20, BBSome protein BBS8 and homodimeric kinesin KIF17, whose function in mammalian cilia is unclear. Interestingly, all five proteins moved at ∼0.45 µm/s in anterograde and retrograde direction, suggesting they are all transported by the same machinery. Moreover, GFP tagged ICK and MOK moved at similar velocities as the IFT proteins, suggesting they are part of, or transported by the IFT machinery. Indeed, loss- or gain-of-function of ICK affected IFT speeds: knockdown increased anterograde velocities, whereas overexpression reduced retrograde speed. In contrast, MOK knockdown or overexpression did not affect IFT speeds. Finally, we found that the effects of ICK or MOK knockdown on cilium length and IFT are suppressed by rapamycin treatment, suggesting that these effects require the mTORC1 pathway. Our results confirm the importance of RCK kinases as regulators of cilium length and IFT. However, whereas some of our results suggest a direct correlation between cilium length and IFT speed, other results indicate that cilium length can be modulated independent of IFT speed. PMID:25243405

Myosin1D is an evolutionarily conserved regulator of animal left-right asymmetry.

PubMed

Juan, Thomas; Géminard, Charles; Coutelis, Jean-Baptiste; Cerezo, Delphine; Polès, Sophie; Noselli, Stéphane; Fürthauer, Maximilian

2018-05-16

The establishment of left-right (LR) asymmetry is fundamental to animal development, but the identification of a unifying mechanism establishing laterality across different phyla has remained elusive. A cilia-driven, directional fluid flow is important for symmetry breaking in numerous vertebrates, including zebrafish. Alternatively, LR asymmetry can be established independently of cilia, notably through the intrinsic chirality of the acto-myosin cytoskeleton. Here, we show that Myosin1D (Myo1D), a previously identified regulator of Drosophila LR asymmetry, is essential for the formation and function of the zebrafish LR organizer (LRO), Kupffer's vesicle (KV). Myo1D controls the orientation of LRO cilia and interacts functionally with the planar cell polarity (PCP) pathway component VanGogh-like2 (Vangl2), to shape a productive LRO flow. Our findings identify Myo1D as an evolutionarily conserved regulator of animal LR asymmetry, and show that functional interactions between Myo1D and PCP are central to the establishment of animal LR asymmetry.

Intraciliary calcium oscillations initiate vertebrate left-right asymmetry.

PubMed

Yuan, Shiaulou; Zhao, Lu; Brueckner, Martina; Sun, Zhaoxia

2015-03-02

Bilateral symmetry during vertebrate development is broken at the left-right organizer (LRO) by ciliary motility and the resultant directional flow of extracellular fluid. However, how ciliary motility is perceived and transduced into asymmetrical intracellular signaling at the LRO remains controversial. Previous work has indicated that sensory cilia and polycystin-2 (Pkd2), a cation channel, are required for sensing ciliary motility, yet their function and the molecular mechanism linking both to left-right signaling cascades are unknown. Here we report novel intraciliary calcium oscillations (ICOs) at the LRO that connect ciliary sensation of ciliary motility to downstream left-right signaling. Utilizing cilia-targeted genetically encoded calcium indicators in live zebrafish embryos, we show that ICOs depend on Pkd2 and are left-biased at the LRO in response to ciliary motility. Asymmetric ICOs occur with onset of LRO ciliary motility, thus representing the earliest known LR asymmetric molecular signal. Suppression of ICOs using a cilia-targeted calcium sink reveals that they are essential for LR development. These findings demonstrate that intraciliary calcium initiates LR development and identify cilia as a functional ion signaling compartment connecting ciliary motility and flow to molecular LR signaling. Copyright © 2015 Elsevier Ltd. All rights reserved.

A study in motion sickness - Saccular hair cells in the adult bullfrog

NASA Technical Reports Server (NTRS)

Cohen, G. M.; Reschke, M.; Homick, J.

1982-01-01

The bullfrog's saccule were examined using light and scanning electron microscopy. No evidence of a striola was found. Type A hair cells were not only distributed peripherally, but also throughout the central macula, though far less frequently than the dominant type D. Two primary hair cell types were distinguished, which corresponded to the ciliary patterns: type A cilia are associated with short, conical hair cells, and type D cilia are associated with long, cylindrical hair cells. Each displays at least one subtype, which may represent developmental precursors. The otolithic membrane is crisscrossed with tunnels and topped with statoconia.

Mutations in the inositol polyphosphate-5-phosphatase E gene link phosphatidyl inositol signaling to the ciliopathies

PubMed Central

Bielas, Stephanie L.; Silhavy, Jennifer L.; Brancati, Francesco; Kisseleva, Marina V.; Al-Gazali, Lihadh; Sztriha, Laszlo; Bayoumi, Riad A.; Zaki, Maha S.; Abdel-Aleem, Alice; Rosti, Ozgur; Kayserili, Hulya; Swistun, Dominika; Scott, Lesley C.; Bertini, Enrico; Boltshauser, Eugen; Fazzi, Elisa; Travaglini, Lorena; Field, Seth J.; Gayral, Stephanie; Jacoby, Monique; Schurmans, Stephane; Dallapiccola, Bruno; Majerus, Philip W.; Valente, Enza Maria; Gleeson, Joseph G.

2009-01-01

Phosphotidylinositol (PtdIns) signaling is tightly regulated, both spatially and temporally, by subcellularly localized PtdIns kinases and phosphatases that dynamically alter downstream signaling events 1. Joubert Syndrome (JS) characterized by a specific midbrain-hindbrain malformation (“molar tooth sign”) and variably associated retinal dystrophy, nephronophthisis, liver fibrosis and polydactyly 2, and is included in the newly emerging group of “ciliopathies”. In patients linking to JBTS1, we identified mutations in the INPP5E gene, encoding inositol polyphosphate-5-phosphatase E, which hydrolyzes the 5-phosphate of PtdIns(3,4,5)P3 and PtdIns(4,5)P2. Mutations clustered in the phosphatase domain and impaired 5-phosphatase activity, resulting in altered cellular PtdIns ratios. INPP5E localized to cilia in major organs affected in JS, and mutations promoted premature destabilization of cilia in response to stimulation. Thus, these data links PtdIns signaling to the primary cilium, a cellular structure that is becoming increasingly appreciated for its role in mediating cell signals and neuronal function. PMID:19668216

Reconstitution reveals motor activation for intraflagellar transport.

PubMed

Mohamed, Mohamed A A; Stepp, Willi L; Ökten, Zeynep

2018-05-01

The human body represents a notable example of ciliary diversification. Extending from the surface of most cells, cilia accomplish a diverse set of tasks. Predictably, mutations in ciliary genes cause a wide range of human diseases such as male infertility and blindness. In Caenorhabditis elegans sensory cilia, this functional diversity appears to be traceable to the differential regulation of the kinesin-2-powered intraflagellar-transport (IFT) machinery. Here we reconstituted the first, to our knowledge, functional multi-component IFT complex that is deployed in the sensory cilia of C. elegans. Our bottom-up approach revealed the molecular basis of specific motor recruitment to the IFT trains. We identified the key component that incorporates homodimeric kinesin-2 into its physiologically relevant context, which in turn allosterically activates the motor for efficient transport. These results will enable the molecular delineation of IFT regulation, which has eluded understanding since its discovery more than two decades ago.

Xenopus Bicaudal-C Is Required for the Differentiation of the Amphibian Pronephros

PubMed Central

Tran, Uyen; Mary Pickney, L.; Duygu Özpolat, B.; Wessely, Oliver

2007-01-01

The RNA-binding molecule Bicaudal-C regulates embryonic development in Drosophila and Xenopus. Interestingly, mouse mutants of Bicaudal-C do not show early patterning defects, but instead develop polycystic kidney disease (PKD). To further investigate the molecular mechanism of Bicaudal-C in kidney development, we analyzed its function in the developing amphibian pronephros. Bicaudal-C mRNA was present in the epithelial structures of the Xenopus pronephros, the tubules and the duct, but not the glomus. Inhibition of the translation of endogenous Bicaudal-C with antisense morpholino oligomers (xBic-C-MO) led to a PKD-like phenotype in Xenopus. Embryos lacking Bicaudal-C developed generalized edemas and dilated pronephric tubules and ducts. This phenotype was caused by impaired differentiation of the pronephros. Molecular markers specifically expressed in the late distal tubule were absent in xBic-C-MO-injected embryos. Furthermore, Bicaudal-C was not required for primary cilia formation, an important organelle affected in PKD. These data support the idea that Bicaudal-C functions downstream or parallel of a cilia-regulated signaling pathway. This pathway is required for terminal differentiation of the late distal tubule of the Xenopus pronephros and regulates renal epithelial cell differentiation, which - when disrupted - results in PKD. PMID:17521625

The mechanics of the primary cilium: an intricate structure with complex function.

PubMed

Hoey, David A; Downs, Matthew E; Jacobs, Christopher R

2012-01-03

The primary cilium is a non-motile singular cellular structure that extends from the surface of nearly every cell in the body. The cilium has been shown to play numerous roles in maintaining tissue homeostasis, through regulating signaling pathways and sensing both biophysical and biochemical changes in the extracellular environment. The structural performance of the cilium is paramount to its function as defective cilia have been linked to numerous pathologies. In particular, the cilium has demonstrated a mechanosensory role in tissues such as the kidney, liver, endothelium and bone, where cilium deflection under mechanical loading triggers a cellular response. Understanding of how cilium structure and subsequent mechanical behavior contributes to the roles that cilium plays in regulating cellular behavior is a compelling question, yet is a relatively untouched research area. Recent advances in biophysical measurements have demonstrated the cilium to be a structurally intricate organelle containing an array of load bearing proteins. Furthermore advances in modeling of this organelle have revealed the importance of these proteins at regulating the cilium's mechanosensitivity. Remarkably, the cilium is capable of adapting its mechanical state, altering its length and possibly it's bending resistance, to regulate its mechanosensitivity demonstrating the importance of cilium mechanics in cellular responses. In this review, we introduce the cilium as a mechanosensor; discuss the advances in the mechanical modeling of cilia; explore the structural features of the cilium, which contribute to its mechanics and finish with possible mechanisms in which alteration in structure may affect ciliary mechanics, consequently affecting ciliary based mechanosensing. Copyright © 2011 Elsevier Ltd. All rights reserved.

Loss-of-Function Mutations in a Human Gene Related to Chlamydomonas reinhardtii Dynein IC78 Result in Primary Ciliary Dyskinesia

PubMed Central

Pennarun, Gaëlle; Escudier, Estelle; Chapelin, Catherine; Bridoux, Anne-Marie; Cacheux, Valère; Roger, Gilles; Clément, Annick; Goossens, Michel; Amselem, Serge; Duriez, Bénédicte

1999-01-01

Summary Primary ciliary dyskinesia (PCD) is a group of heterogeneous disorders of unknown origin, usually inherited as an autosomal recessive trait. Its phenotype is characterized by axonemal abnormalities of respiratory cilia and sperm tails leading to bronchiectasis and sinusitis, which are sometimes associated with situs inversus (Kartagener syndrome) and male sterility. The main ciliary defect in PCD is an absence of dynein arms. We have isolated the first gene involved in PCD, using a candidate-gene approach developed on the basis of documented abnormalities of immotile strains of Chlamydomonas reinhardtii, which carry axonemal ultrastructural defects reminiscent of PCD. Taking advantage of the evolutionary conservation of genes encoding axonemal proteins, we have isolated a human sequence (DNAI1) related to IC78, a C. reinhardtii gene encoding a dynein intermediate chain in which mutations are associated with the absence of outer dynein arms. DNAI1 is highly expressed in trachea and testis and is composed of 20 exons located at 9p13-p21. Two loss-of-function mutations of DNAI1 have been identified in a patient with PCD characterized by immotile respiratory cilia lacking outer dynein arms. In addition, we excluded linkage between this gene and similar PCD phenotypes in five other affected families, providing a clear demonstration of locus heterogeneity. These data reveal the critical role of DNAI1 in the development of human axonemal structures and open up new means for identification of additional genes involved in related developmental defects. PMID:10577904

Sept7b is essential for pronephric function and development of left-right asymmetry in zebrafish embryogenesis.

PubMed

Dash, Surjya Narayan; Lehtonen, Eero; Wasik, Anita A; Schepis, Antonino; Paavola, Jere; Panula, Pertti; Nelson, W James; Lehtonen, Sanna

2014-04-01

The conserved septin family of filamentous small GTPases plays important roles in mitosis, cell migration and cell morphogenesis by forming scaffolds and diffusion barriers. Recent studies in cultured cells in vitro indicate that a septin complex of septin 2, 7 and 9 is required for ciliogenesis and cilia function, but septin function in ciliogenesis in vertebrate organs in vivo is not understood. We show that sept7b is expressed in ciliated cells in different tissues during early zebrafish development. Knockdown of sept7b by using morpholino antisense oligonucleotides caused misorientation of basal bodies and cilia, reduction of apical actin and the shortening of motile cilia in Kupffer's vesicle and pronephric tubules. This resulted in pericardial and yolk sac edema, body axis curvature and hydrocephaly. Notably, in sept7b morphants we detected strong left-right asymmetry defects in the heart and lateral plate mesoderm (situs inversus), reduced fluid flow in the kidney, the formation of kidney cysts and loss of glomerular filtration barrier function. Thus, sept7b is essential during zebrafish development for pronephric function and ciliogenesis, and loss of expression of sept7b results in defects that resemble human ciliopathies.

Value of transmission electron microscopy for primary ciliary dyskinesia diagnosis in the era of molecular medicine: Genetic defects with normal and non-diagnostic ciliary ultrastructure.

PubMed

Shapiro, Adam J; Leigh, Margaret W

2017-01-01

Primary ciliary dyskinesia (PCD) is a genetic disorder causing chronic oto-sino-pulmonary disease. No single diagnostic test will detect all PCD cases. Transmission electron microscopy (TEM) of respiratory cilia was previously considered the gold standard diagnostic test for PCD, but 30% of all PCD cases have either normal ciliary ultrastructure or subtle changes which are non-diagnostic. These cases are identified through alternate diagnostic tests, including nasal nitric oxide measurement, high-speed videomicroscopy analysis, immunofluorescent staining of axonemal proteins, and/or mutation analysis of various PCD causing genes. Autosomal recessive mutations in DNAH11 and HYDIN produce normal TEM ciliary ultrastructure, while mutations in genes encoding for radial spoke head proteins result in some cross-sections with non-diagnostic alterations in the central apparatus interspersed with normal ciliary cross-sections. Mutations in nexin link and dynein regulatory complex genes lead to a collection of different ciliary ultrastructures; mutations in CCDC65, CCDC164, and GAS8 produce normal ciliary ultrastructure, while mutations in CCDC39 and CCDC40 cause absent inner dynein arms and microtubule disorganization in some ciliary cross-sections. Mutations in CCNO and MCIDAS cause near complete absence of respiratory cilia due to defects in generation of multiple cellular basal bodies; however, the scant cilia generated may have normal ultrastructure. Lastly, a syndromic form of PCD with retinal degeneration results in normal ciliary ultrastructure through mutations in the RPGR gene. Clinicians must be aware of these genetic causes of PCD resulting in non-diagnostic TEM ciliary ultrastructure and refrain from using TEM of respiratory cilia as a test to rule out PCD.

Quantification and visualization of injury and regeneration in the developing ciliated epithelium using quantitative flow imaging and speckle variance optical coherence tomography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Gamm, Ute A.; Huang, Brendan K.; Mis, Emily K.; Khokha, Mustafa K.; Choma, Michael A.

2017-02-01

Premature infants are at a high risk for respiratory diseases owing to an underdeveloped respiratory system that is very susceptible to infection and inflammation. One aspect of respiratory health is the state of the ciliated respiratory epithelium which lines the trachea and bronchi. The ciliated epithelium is responsible for trapping and removing pathogens and pollutants from the lungs and an impairment of ciliary functionality can lead to recurring respiratory infections and subsequent lung damage. Mechanisms of cilia-driven fluid flow itself but also factors influenced by development like ciliary density and flow generation are incompletely understood. Furthermore, medical interventions like intubation and accidental aspiration can lead to focal or diffuse loss of cilia and disruption of flow. In this study we use two animal models, Xenopus embryo and ex vivo mouse trachea, to analyze flow defects in the injured ciliated epithelium. Injury is generated either mechanically with a scalpel or chemically by calcium chloride (CaCl2) shock, which efficiently but reversibly deciliates the embryo skin. In this study we used optical coherence tomography (OCT) and particle tracking velocimetry (PTV) to quantify cilia driven fluid flow over the surface of the Xenopus embryo. We additionally visualized damage to the ciliated epithelium by capturing 3D speckle variance images that highlight beating cilia. Mechanical injury disrupted cilia-driven fluid flow over the injured site, which led to a reduction in cilia-driven fluid flow over the whole surface of the embryo (n=7). The calcium chloride shock protocol proved to be highly effective in deciliating embryos (n=6). 3D speckle variance images visualized a loss of cilia and cilia-driven flow was halted immediately after application. We also applied CaCl2-shock to cultured ex vivo mouse trachea (n=8) and found, similarly to effects in Xenopus embryo, an extensive loss of cilia with resulting cessation of flow. We investigated the regeneration of the ciliated epithelium after an 8 day incubation period, and found that cilia had regrown and flow was completely restored. In conclusion, OCT is a valuable tool to visualize injury of the ciliated epithelium and to quantify reduction of generated flow. This method allows for systematic investigation of focal and diffuse injury of the ciliated epithelium and the assessment of mechanisms to compensate for loss of flow.

Metachronal Motion of Artificial Magnetic Cilia

NASA Astrophysics Data System (ADS)

Hanasoge, Srinivas; Hesketh, Peter; Alexeev, Alexander

2017-11-01

Most microorganisms use asymmetrically oscillating hair like cilia on their surface to achieve fluid transport. These cilia are often seen to beat in a metachronal fashion with a constant phase difference with the neighbors which generates a travelling wave. Although the origin of metachronal waves in such cilia is not well understood, mimicking such behavior in synthetic systems could prove useful in achieving similar advantages. In this work, we demonstrate metachronal waves in synthetic magnetic ciliary systems. The soft magnetic cilia are forced by a uniform rotating magnetic field. The cilia bend as the field rotates and tend to align along the direction of field to minimize the potential energy. Longer cilia bend to a larger degree, while the shorter cilia show less bending. This difference in the bending of cilia based on their length leads to a phase difference in their oscillation cycle. We exploit this phase differences to metachronally oscillate the synthetic cilia. We fabricate an array consisting of cilia with increasing lengths, in which the cilia beat with a constant phase difference with the neighboring cilia, producing a travelling wave. Such behavior could potentially be useful in enhanced fluid and particle transport as seen in natural systems. USDA.

Interkinetic nuclear migration in the mouse embryonic ureteric epithelium: Possible implication for congenital anomalies of the kidney and urinary tract.

PubMed

Motoya, Tomoyuki; Ogawa, Noriko; Nitta, Tetsuya; Rafiq, Ashiq Mahmood; Jahan, Esrat; Furuya, Motohide; Matsumoto, Akihiro; Udagawa, Jun; Otani, Hiroki

2016-05-01

Interkinetic nuclear migration (INM) is a phenomenon in which progenitor cell nuclei migrate along the apico-basal axis of the pseudostratified epithelium, which is characterized by the presence of apical primary cilia, in synchrony with the cell cycle in a manner of apical mitosis. INM is suggested to regulate not only stem/progenitor cell proliferation/differentiation but also organ size and shape. INM has been reported in epithelia of both ectoderm and endoderm origin. We examined whether INM exists in the mesoderm-derived ureteric epithelium. At embryonic day (E) 11.5, E12.5 and E13.5, C57BL/6J mouse dams were injected with 5-bromo-2'-deoxyuridine (BrdU) and embryos were killed 1, 2, 4, 6, 8, 10 and 12 h later. We immunostained transverse sections of the ureter for BrdU, and measured the position of BrdU (+) nuclei in the ureteric epithelia along the apico-basal axis at each time point. We analyzed the distribution patterns of BrdU (+) nuclei in histograms using the multidimensional scaling. Changes in the nucleus distribution patterns suggested nucleus movement characteristic of INM in the ureteric epithelia, and the mode of INM varied throughout the ureter development. While apical primary cilia are related with INM by providing a centrosome for the apical mitosis, congenital anomalies of the kidney and urinary tract (CAKUT) include syndromes linked to primary ciliary dysfunction affecting epithelial tubular organs such as kidney, ureter, and brain. The present study showed that INM exists in the ureteric epithelium and suggests that INM may be related with the CAKUT etiology via primary ciliary protein function. © 2015 Japanese Teratology Society.

Voltage-gated calcium channels of Paramecium cilia

PubMed Central

Lodh, Sukanya; Valentine, Megan S.; Van Houten, Judith L.

2016-01-01

ABSTRACT Paramecium cells swim by beating their cilia, and make turns by transiently reversing their power stroke. Reversal is caused by Ca2+ entering the cilium through voltage-gated Ca2+ (CaV) channels that are found exclusively in the cilia. As ciliary Ca2+ levels return to normal, the cell pivots and swims forward in a new direction. Thus, the activation of the CaV channels causes cells to make a turn in their swimming paths. For 45 years, the physiological characteristics of the Paramecium ciliary CaV channels have been known, but the proteins were not identified until recently, when the P. tetraurelia ciliary membrane proteome was determined. Three CaVα1 subunits that were identified among the proteins were cloned and confirmed to be expressed in the cilia. We demonstrate using RNA interference that these channels function as the ciliary CaV channels that are responsible for the reversal of ciliary beating. Furthermore, we show that Pawn (pw) mutants of Paramecium that cannot swim backward for lack of CaV channel activity do not express any of the three CaV1 channels in their ciliary membrane, until they are rescued from the mutant phenotype by expression of the wild-type PW gene. These results reinforce the correlation of the three CaV channels with backward swimming through ciliary reversal. The PwB protein, found in endoplasmic reticulum fractions, co-immunoprecipitates with the CaV1c channel and perhaps functions in trafficking. The PwA protein does not appear to have an interaction with the channel proteins but affects their appearance in the cilia. PMID:27707864

Nature-inspired micro-fluidic manipulation using artificial cilia

NASA Astrophysics Data System (ADS)

den Toonder, Jaap; de Goede, Judith; Khatavkar, Vinayak; Anderson, Patrick

2006-11-01

One particular micro-fluidics manipulation mechanism ``designed'' by nature is that due to a covering of beating cilia over the external surface of micro-organisms (e.g. Paramecium). A cilium can be viewed as a small hair or flexible rod (in protozoa: typical length 10 μm and diameter 0.1 μm) which is attached to the surface. We have developed polymer micro-actuators, made with standard micro-technology processing, which respond to an applied electrical or magnetic field by changing their shape. The shape and size of the polymer actuators mimics that of cilia occurring in nature. We have shown experimentally that, indeed, our artificial cilia can induce significant flow velocities of at least 75 μm/s in a fluid with a viscosity of 10 mPas. In this paper we will give an overview of our activities in developing the polymer actuators and the corresponding technology, show experimental and numerical fluid flow results, and finally assess the feasibility of applying this new and attractive micro-fluidic actuation method in functional biosensors.

dTULP, the Drosophila melanogaster Homolog of Tubby, Regulates Transient Receptor Potential Channel Localization in Cilia

PubMed Central

Shim, Jaewon; Han, Woongsu; Lee, Jinu; Bae, Yong Chul; Chung, Yun Doo; Kim, Chul Hoon; Moon, Seok Jun

2013-01-01

Mechanically gated ion channels convert sound into an electrical signal for the sense of hearing. In Drosophila melanogaster, several transient receptor potential (TRP) channels have been implicated to be involved in this process. TRPN (NompC) and TRPV (Inactive) channels are localized in the distal and proximal ciliary zones of auditory receptor neurons, respectively. This segregated ciliary localization suggests distinct roles in auditory transduction. However, the regulation of this localization is not fully understood. Here we show that the Drosophila Tubby homolog, King tubby (hereafter called dTULP) regulates ciliary localization of TRPs. dTULP-deficient flies show uncoordinated movement and complete loss of sound-evoked action potentials. Inactive and NompC are mislocalized in the cilia of auditory receptor neurons in the dTulp mutants, indicating that dTULP is required for proper cilia membrane protein localization. This is the first demonstration that dTULP regulates TRP channel localization in cilia, and suggests that dTULP is a protein that regulates ciliary neurosensory functions. PMID:24068974

Circulation of fluids in the gastrovascular system of a stoloniferan octocoral.

PubMed

Parrin, Austin P; Netherton, Sarah E; Bross, Lori S; McFadden, Catherine S; Blackstone, Neil W

2010-10-01

Cilia-based transport systems characterize sponges and placozoans. Cilia are employed in cnidarian gastrovascular systems as well, but typically function in concert with muscular contractions. Previous reports suggest that anthozoans may be an exception to this pattern, utilizing only cilia in their gastrovascular systems. With an inverted microscope and digital image analysis, we used stoloniferan octocoral colonies growing on microscope cover glass to quantitatively describe the movement of fluids in this system for the first time. Flow in stolons (diameter ≈300 μm) is simultaneously bidirectional, with average velocities of 100-200 μm/s in each direction. Velocities are maximal immediately adjacent to the stolon wall and decrease to a minimum in the center of the stolon. Flow velocity is unaffected by stolonal contractions, suggesting that muscular peristalsis is not a factor in propelling the flow. Stolon intersections (diameter ≈500 μm) occur below polyps and serve as traffic roundabouts with unidirectional, circular flow. Such cilia-driven transport may be the plesiomorphic state for the gastrovascular system of cnidarians.

Regulation of Polycystin-1 Function by Calmodulin Binding

PubMed Central

Doerr, Nicholas; Wang, Yidi; Kipp, Kevin R.; Liu, Guangyi; Benza, Jesse J.; Pletnev, Vladimir; Pavlov, Tengis S.; Staruschenko, Alexander; Mohieldin, Ashraf M.; Takahashi, Maki; Nauli, Surya M.; Weimbs, Thomas

2016-01-01

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common genetic disease that leads to progressive renal cyst growth and loss of renal function, and is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The PC1/PC2 complex localizes to primary cilia and can act as a flow-dependent calcium channel in addition to numerous other signaling functions. The exact functions of the polycystins, their regulation and the purpose of the PC1/PC2 channel are still poorly understood. PC1 is an integral membrane protein with a large extracytoplasmic N-terminal domain and a short, ~200 amino acid C-terminal cytoplasmic tail. Most proteins that interact with PC1 have been found to bind via the cytoplasmic tail. Here we report that the PC1 tail has homology to the regulatory domain of myosin heavy chain including a conserved calmodulin-binding motif. This motif binds to CaM in a calcium-dependent manner. Disruption of the CaM-binding motif in PC1 does not affect PC2 binding, cilia targeting, or signaling via heterotrimeric G-proteins or STAT3. However, disruption of CaM binding inhibits the PC1/PC2 calcium channel activity and the flow-dependent calcium response in kidney epithelial cells. Furthermore, expression of CaM-binding mutant PC1 disrupts cellular energy metabolism. These results suggest that critical functions of PC1 are regulated by its ability to sense cytosolic calcium levels via binding to CaM. PMID:27560828

Functional Redundancy of the B9 Proteins and Nephrocystins in Caenorhabditis elegans Ciliogenesis

PubMed Central

Williams, Corey L.; Winkelbauer, Marlene E.; Schafer, Jenny C.; Michaud, Edward J.

2008-01-01

Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and Joubert syndrome (JBTS) are a group of heterogeneous cystic kidney disorders with partially overlapping loci. Many of the proteins associated with these diseases interact and localize to cilia and/or basal bodies. One of these proteins is MKS1, which is disrupted in some MKS patients and contains a B9 motif of unknown function that is found in two other mammalian proteins, B9D2 and B9D1. Caenorhabditis elegans also has three B9 proteins: XBX-7 (MKS1), TZA-1 (B9D2), and TZA-2 (B9D1). Herein, we report that the C. elegans B9 proteins form a complex that localizes to the base of cilia. Mutations in the B9 genes do not overtly affect cilia formation unless they are in combination with a mutation in nph-1 or nph-4, the homologues of human genes (NPHP1 and NPHP4, respectively) that are mutated in some NPHP patients. Our data indicate that the B9 proteins function redundantly with the nephrocystins to regulate the formation and/or maintenance of cilia and dendrites in the amphid and phasmid ciliated sensory neurons. Together, these data suggest that the human homologues of the novel B9 genes B9D2 and B9D1 will be strong candidate loci for pathologies in human MKS, NPHP, and JBTS. PMID:18337471

Directed Fluid Transport and Mixing with Biomimetic Cilia Arrays

NASA Astrophysics Data System (ADS)

Shields, A. R.; Evans, B. A.; Carstens, B. L.; Falvo, M. R.; Washburn, S.; Superfine, R.

2009-03-01

We present results on the long-range, directed fluid transport and fluidic mixing produced by the collective beating of arrays of biomimetic cilia. These artificial cilia are arrays of free-standing nanorods roughly the size of biological cilia, which we fabricate from a polymer-magnetic nanoparticle composite material and actuate with permanent magnets to mimic biological cilia. Biological cilia have evolved to produce microscale fluid transport and are increasingly being recognized as critical components in a wide range of biological systems. However, despite much effort cilia generated fluid flows remain an area of active study. In the last decade, cilia-driven fluid flow in the embryonic node of vertebrates has been implicated as the initial left-right symmetry breaking event in these embryos. With silia we generate directional fluid transport by mimicking the tilted conical beating of these nodal cilia. By seeding fluorescent microparticles into the fluid we have noted the existence of two distinct flow regimes. The fluid flow is directional and coherent above the cilia tips, while between the cilia tips and the floor particle motion is complicated and suggestive of chaotic advection.

Dynamics of Individual cilia to external loading- A simple one dimensional picture

NASA Astrophysics Data System (ADS)

Swaminathan, Vinay; Hill, David; Superfine, R.

2008-10-01

From being called the cellular janitors to swinging debauchers, cilia have captured the fascinations of researchers for over 200 years. In cystic fibrosis and chronic obstructive pulmonary disease where the cilia loses it's function, the protective mucus layer in the lung thickens and mucociliary clearance breaks down, leading to inflammation along the airways and an increased rate of infection. The mechanistic understanding of mucus clearance depends on a quantitative assessment of the axoneme dynamics and the maximum force the cilia are capable of generating and imparting to the mucus layer. Similar to the situation in molecular motors, detailed quantitative measurements of dynamics under applied load conditions are expected to be essential in developing predictive models. Based on our measurements of the dynamics of individual ciliary motion in the human bronchial epithelial cell under the application of an applied load, we present a simple one dimensional model for the axoneme dynamics and quantify the axoneme stiffness, the internal force generated by the axoneme, the stall force and show how the dynamics sheds insight on the time dependence of the internal force generation. The internal force generated by the axoneme is related to the ability of cilia to propel fluids and to their potential role in force sensing.

Functional architecture of olfactory ionotropic glutamate receptors.

PubMed

Abuin, Liliane; Bargeton, Benoîte; Ulbrich, Maximilian H; Isacoff, Ehud Y; Kellenberger, Stephan; Benton, Richard

2011-01-13

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate chemical communication between neurons at synapses. A variant iGluR subfamily, the Ionotropic Receptors (IRs), was recently proposed to detect environmental volatile chemicals in olfactory cilia. Here, we elucidate how these peripheral chemosensors have evolved mechanistically from their iGluR ancestors. Using a Drosophila model, we demonstrate that IRs act in combinations of up to three subunits, comprising individual odor-specific receptors and one or two broadly expressed coreceptors. Heteromeric IR complex formation is necessary and sufficient for trafficking to cilia and mediating odor-evoked electrophysiological responses in vivo and in vitro. IRs display heterogeneous ion conduction specificities related to their variable pore sequences, and divergent ligand-binding domains function in odor recognition and cilia localization. Our results provide insights into the conserved and distinct architecture of these olfactory and synaptic ion channels and offer perspectives into the use of IRs as genetically encoded chemical sensors. Copyright © 2011 Elsevier Inc. All rights reserved.

Drosophila PLP assembles pericentriolar clouds that promote centriole stability, cohesion and MT nucleation.

PubMed

Roque, Helio; Saurya, Saroj; Pratt, Metta B; Johnson, Errin; Raff, Jordan W

2018-02-01

Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. It has been implicated in many aspects of centrosome and cilia function, but its precise role is unclear. Here, we examine Drosophila Pericentrin-like-protein (PLP) function in vivo in tissues that form both centrosomes and cilia. Plp mutant centrioles exhibit four major defects: (1) They are short and have subtle structural abnormalities; (2) They disengage prematurely, and so overduplicate; (3) They organise fewer cytoplasmic MTs during interphase; (4) When forming cilia, they fail to establish and/or maintain a proper connection to the plasma membrane-although, surprisingly, they can still form an axoneme-like structure that can recruit transition zone (TZ) proteins. We show that PLP helps assemble "pericentriolar clouds" of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. We propose that the partial loss of these structures may largely explain the complex centriole, centrosome and cilium defects we observe in Plp mutant cells.

Voltage-gated calcium channels of Paramecium cilia.

PubMed

Lodh, Sukanya; Yano, Junji; Valentine, Megan S; Van Houten, Judith L

2016-10-01

Paramecium cells swim by beating their cilia, and make turns by transiently reversing their power stroke. Reversal is caused by Ca 2+ entering the cilium through voltage-gated Ca 2+ (Ca V ) channels that are found exclusively in the cilia. As ciliary Ca 2+ levels return to normal, the cell pivots and swims forward in a new direction. Thus, the activation of the Ca V channels causes cells to make a turn in their swimming paths. For 45 years, the physiological characteristics of the Paramecium ciliary Ca V channels have been known, but the proteins were not identified until recently, when the P. tetraurelia ciliary membrane proteome was determined. Three Ca V α1 subunits that were identified among the proteins were cloned and confirmed to be expressed in the cilia. We demonstrate using RNA interference that these channels function as the ciliary Ca V channels that are responsible for the reversal of ciliary beating. Furthermore, we show that Pawn (pw) mutants of Paramecium that cannot swim backward for lack of Ca V channel activity do not express any of the three Ca V 1 channels in their ciliary membrane, until they are rescued from the mutant phenotype by expression of the wild-type PW gene. These results reinforce the correlation of the three Ca V channels with backward swimming through ciliary reversal. The PwB protein, found in endoplasmic reticulum fractions, co-immunoprecipitates with the Ca V 1c channel and perhaps functions in trafficking. The PwA protein does not appear to have an interaction with the channel proteins but affects their appearance in the cilia. © 2016. Published by The Company of Biologists Ltd.

The Meckel syndrome- associated protein MKS1 functionally interacts with components of the BBSome and IFT complexes to mediate ciliary trafficking and hedgehog signaling

PubMed Central

Barrington, Chloe L.; Katsanis, Nicholas

2017-01-01

The importance of primary cilia in human health is underscored by the link between ciliary dysfunction and a group of primarily recessive genetic disorders with overlapping clinical features, now known as ciliopathies. Many of the proteins encoded by ciliopathy-associated genes are components of a handful of multi-protein complexes important for the transport of cargo to the basal body and/or into the cilium. A key question is whether different complexes cooperate in cilia formation, and whether they participate in cilium assembly in conjunction with intraflagellar transport (IFT) proteins. To examine how ciliopathy protein complexes might function together, we have analyzed double mutants of an allele of the Meckel syndrome (MKS) complex protein MKS1 and the BBSome protein BBS4. We find that Mks1; Bbs4 double mutant mouse embryos exhibit exacerbated defects in Hedgehog (Hh) dependent patterning compared to either single mutant, and die by E14.5. Cells from double mutant embryos exhibit a defect in the trafficking of ARL13B, a ciliary membrane protein, resulting in disrupted ciliary structure and signaling. We also examined the relationship between the MKS complex and IFT proteins by analyzing double mutant between Mks1 and a hypomorphic allele of the IFTB component Ift172. Despite each single mutant surviving until around birth, Mks1; Ift172avc1 double mutants die at mid-gestation, and exhibit a dramatic failure of cilia formation. We also find that Mks1 interacts genetically with an allele of Dync2h1, the IFT retrograde motor. Thus, we have demonstrated that the MKS transition zone complex cooperates with the BBSome to mediate trafficking of specific trans-membrane receptors to the cilium. Moreover, the genetic interaction of Mks1 with components of IFT machinery suggests that the transition zone complex facilitates IFT to promote cilium assembly and structure. PMID:28291807

A common variant in combination with a nonsense mutation in a member of the thioredoxin family causes primary ciliary dyskinesia

PubMed Central

Duriez, Bénédicte; Duquesnoy, Philippe; Escudier, Estelle; Bridoux, Anne-Marie; Escalier, Denise; Rayet, Isabelle; Marcos, Elisabeth; Vojtek, Anne-Marie; Bercher, Jean-François; Amselem, Serge

2007-01-01

Thioredoxins belong to a large family of enzymatic proteins that function as general protein disulfide reductases, therefore participating in several cellular processes via redox-mediated reactions. So far, none of the 18 members of this family has been involved in human pathology. Here we identified TXNDC3, which encodes a thioredoxin–nucleoside diphosphate kinase, as a gene implicated in primary ciliary dyskinesia (PCD), a genetic condition characterized by chronic respiratory tract infections, left–right asymmetry randomization, and male infertility. We show that the disease, which segregates as a recessive trait, results from the unusual combination of the following two transallelic defects: a nonsense mutation and a common intronic variant found in 1% of control chromosomes. This variant affects the ratio of two physiological TXNDC3 transcripts: the full-length isoform and a novel isoform, TXNDC3d7, carrying an in-frame deletion of exon 7. In vivo and in vitro expression data unveiled the physiological importance of TXNDC3d7 (whose expression was reduced in the patient) and the corresponding protein that was shown to bind microtubules. PCD is known to result from defects of the axoneme, an organelle common to respiratory cilia, embryonic nodal cilia, and sperm flagella, containing dynein arms, with, to date, the implication of genes encoding dynein proteins. Our findings, which identify a another class of molecules involved in PCD, disclose the key role of TXNDC3 in ciliary function; they also point to an unusual mechanism underlying a Mendelian disorder, which is an SNP-induced modification of the ratio of two physiological isoforms generated by alternative splicing. PMID:17360648

Paramecia swimming in viscous flow

NASA Astrophysics Data System (ADS)

Zhang, P.; Jana, S.; Giarra, M.; Vlachos, P. P.; Jung, S.

2015-12-01

Ciliates like Paramecia exhibit fore-aft asymmetry in their body shapes, and preferentially swim in the direction of the slender anterior rather than the wider posterior. However, the physical reasons for this preference are not well understood. In this work, we propose that specific features of the fluid flow around swimming Paramecia confer some energetic advantage to the preferred swimming direction. Therefore, we seek to understand the effects of body asymmetry and swimming direction on the efficiency of swimming and the flux of fluid into the cilia layer (and thus of food into the oral groove), which we assumed to be primary factors in the energy budgets of these organisms. To this end, we combined numerical techniques (the boundary element method) and laboratory experiments (micro particle image velocimetry) to develop a quantitative model of the flow around a Paramecium and investigate the effect of the body shape on the velocity fields, as well as on the swimming and feeding behaviors. Both simulation and experimental results show that velocity fields exhibit fore-aft asymmetry. Moreover, the shape asymmetry revealed an increase of the fluid flux into the cilia layer compared to symmetric body shapes. Under the assumption that cilia fluid intake and feeding efficiency are primary factors in the energy budgets of Paramecia, our model predicts that the anterior swimming direction is energetically favorable to the posterior swimming direction.

Study of heat transfer on physiological driven movement with CNT nanofluids and variable viscosity.

PubMed

Akbar, Noreen Sher; Kazmi, Naeem; Tripathi, Dharmendra; Mir, Nazir Ahmed

2016-11-01

With ongoing interest in CNT nanofluids and materials in biotechnology, energy and environment, microelectronics, composite materials etc., the current investigation is carried out to analyze the effects of variable viscosity and thermal conductivity of CNT nanofluids flow driven by cilia induced movement through a circular cylindrical tube. Metachronal wave is generated by the beating of cilia and mathematically modeled as elliptical wave propagation by Blake (1971). The problem is formulated in the form of nonlinear partial differential equations, which are simplified by using the dimensional analysis to avoid the complicacy of dimensional homogeneity. Lubrication theory is employed to linearize the governing equations and it is also physically appropriate for cilia movement. Analytical solutions for velocity, temperature and pressure gradient and stream function are obtained. The analytical results are numerically simulated by using the Mathematica Software and plotted the graphs for velocity profile, temperature profile, pressure gradient and stream lines for better discussion and visualization. This model is applicable in physiological transport phenomena to explore the nanotechnology in engineering the artificial cilia and ciliated tube/pipe. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Three types of ependymal cells with intracellular calcium oscillation are characterized by distinct cilia beating properties.

PubMed

Liu, Tongyu; Jin, Xingjian; Prasad, Rahul M; Sari, Youssef; Nauli, Surya M

2014-09-01

Ependymal cells are multiciliated epithelial cells that line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia has been associated with various neurological deficits. For the first time, we report three distinct ependymal cell types, I, II, and III, based on their unique ciliary beating frequency and beating angle. These ependymal cells have specific localizations within the third ventricle of the mouse brain. Furthermore, neither ependymal cell types nor their localizations are altered by aging. Our high-speed fluorescence imaging analysis reveals that these ependymal cells have an intracellular pacing calcium oscillation property. Our study further shows that alcohol can significantly repress the amplitude of calcium oscillation and the frequency of ciliary beating, resulting in an overall decrease in volume replacement by the cilia. Furthermore, the pharmacological agent cilostazol could differentially increase cilia beating frequency in type II, but not in type I or type III, ependymal cells. In summary, we provide the first evidence of three distinct types of ependymal cells with calcium oscillation properties. © 2014 Wiley Periodicals, Inc.

A healthy understanding of intraflagellar transport.

PubMed

Sloboda, Roger D

2002-05-01

A microtubule-dependent motility process called intraflagellar transport (IFT) occurs beneath the plasma membrane of cilia and flagella. IFT was first observed in Chlamydomonas, and orthologs of some of the polypeptides involved in IFT have recently been identified in other organisms, including C. elegans and the mouse. In addition to a role in the assembly and maintenance of cilia and flagella, evidence is reviewed here that indicates defects in the process of IFT may be related to problems with human health. Moreover, recent data suggest the possibility of two new roles for IFT in cell function. The first is in transcriptional control of the genes encoding ciliary and flagellar proteins. IFT could provide a mechanism whereby the cell senses the presence or absence of its cilia or flagella and responds by turning on gene transcription resulting in replacement of the missing organelle. The second role is in signal transduction, whereby cilia act as sensors of the external cellular environment and transduce information about the surroundings into intracellular signals that are sent via IFT to the cell body, thus inducing an appropriate cellular response to the environment. Copyright 2002 Wiley-Liss, Inc.

Kin5 Knockdown in Tetrahymena thermophila Using RNAi Blocks Cargo Transport of Gef1

PubMed Central

Awan, Aashir; Bell, Aaron J.; Satir, Peter

2009-01-01

A critical process that builds and maintains the eukaryotic cilium is intraflagellar transport (IFT). This process utilizes members of the kinesin-2 superfamily to transport cargo into the cilium (anterograde transport) and a dynein motor for the retrograde traffic. Using a novel RNAi knockdown method, we have analyzed the function of the homodimeric IFT kinesin-2, Kin5, in Tetrahymena ciliary transport. In RNAi transformants, Kin5 was severely downregulated and disappeared from the cilia, but cilia did not resorb, although tip structure was affected. After deciliation of the knockdown cell, cilia regrew and cells swam, which suggested that Kin5 is not responsible for the trafficking of axonemal precursors to build the cilium, but could be transporting molecules that act in ciliary signal transduction, such as guanine nucleotide exchange proteins (GEFs). Gef1 is a Tetrahymena ciliary protein, and current coimmunoprecipitation and immunofluorescence studies showed that it is absent in regrowing cilia of the knockdown cells lacking ciliary Kin5. We suggest that one important cargo of Kin5 is Gef1 and knockdown of Kin5 results in cell lethality. PMID:19290045

Revealing the Molecular Structure and the Transport Mechanism at the Base of Primary Cilia Using Superresolution STED Microscopy

NASA Astrophysics Data System (ADS)

Yang, Tung-Lin

The primary cilium is an organelle that serves as a signaling center of the cell and is involved in the hedgehog signaling, cAMP pathway, Wnt pathways, etc. Ciliary function relies on the transportation of molecules between the primary cilium and the cell, which is facilitated by intraflagellar transport (IFT). IFT88, one of the important IFT proteins in complex B, is known to play a role in the formation and maintenance of cilia in various types of organisms. The ciliary transition zone (TZ), which is part of the gating apparatus at the ciliary base, is home to a large number of ciliopathy molecules. Recent studies have identified important regulating elements for TZ gating in cilia. However, the architecture of the TZ region and its arrangement relative to intraflagellar transport (IFT) proteins remain largely unknown, hindering the mechanistic understanding of the regulation processes. One of the major challenges comes from the tiny volume at the ciliary base packed with numerous proteins, with the diameter of the TZ close to the diffraction limit of conventional microscopes. Using a series of stimulated emission depletion (STED) superresolution images mapped to electron microscopy images, we analyzed the structural organization of the ciliary base. Subdiffraction imaging of TZ components defines novel geometric distributions of RPGRIP1L, MKS1, CEP290, TCTN2 and TMEM67, shedding light on their roles in TZ structure, assembly, and function. We found TCTN2 at the outmost periphery of the TZ close to the ciliary membrane, with a 227+/-18 nm diameter. TMEM67 was adjacent to TCTN2, with a 205+/-20 nm diameter. RPGRIP1L was localized toward the axoneme at the same axial level as TCTN2 and TMEM67, with a 165+/-8 nm diameter. MKS1 was situated between TMEM67 and RPGRIP1L, with an 186+/-21 nm diameter. Surprisingly, CEP290 was localized at the proximal side of the TZ close to the distal end of the centrin-labeled basal body. The lateral width was unexpectedly close to the width of the basal body, distant from the potential Y-links region of the TZ. Moreover, IFT88 was intriguingly distributed in two distinct patterns, forming three puncta or a Y shape at the ciliary base found in human retinal pigment epithelial cells (RPE), human fibroblasts (HFF), mouse inner medullary collecting duct (IMCD) cells and mouse embryonic fibroblasts (MEFs). We hypothesize that the two distribution states of IFT88 correspond to the open and closed gating states of the TZ, where IFT particles aggregate to form three puncta when the gate is closed, and move to form the branches of the Y-shape pattern when the gate is open. Two reservoirs of IFT particles, correlating with phases of ciliary growth, were localized relative to the internal structure of the TZ. These subdiffraction images reveal unprecedented architectural details of the TZ, providing a basic structural framework for future functional studies. To visualize the dynamic movement of IFT particles within primary cilia, we further conducted superresolution live-cell imaging of IFT88 fused to EYFP in IMCD cells. Our findings, in particular, show IFT88 particles pass through the TZ at a reduced speed by approximately 50%, implying the gating mechanism is involved at this region to slow down IFT trafficking. Finally, we report the distinct transport pathways of IFT88 and Smo (Smoothened), an essential player to hedgehog signaling, to support our hypothesis that two proteins are transported in different mechanisms at the ciliary base, based on dual-color superresolution imaging.

Magnetically-actuated artificial cilia for microfluidic propulsion.

PubMed

Khaderi, S N; Craus, C B; Hussong, J; Schorr, N; Belardi, J; Westerweel, J; Prucker, O; Rühe, J; den Toonder, J M J; Onck, P R

2011-06-21

In this paper we quantitatively analyse the performance of magnetically-driven artificial cilia for lab-on-a-chip applications. The artificial cilia are fabricated using thin polymer films with embedded magnetic nano-particles and their deformation is studied under different external magnetic fields and flows. A coupled magneto-mechanical solid-fluid model that accurately captures the interaction between the magnetic field, cilia and fluid is used to simulate the cilia motion. The elastic and magnetic properties of the cilia are obtained by fitting the results of the computational model to the experimental data. The performance of the artificial cilia with a non-uniform cross-section is characterised using the numerical model for two channel configurations that are of practical importance: an open-loop and a closed-loop channel. We predict that the flow and pressure head generated by the artificial cilia can be as high as 18 microlitres per minute and 3 mm of water, respectively. We also study the effect of metachronal waves on the flow generated and show that the fluid propelled increases drastically compared to synchronously beating cilia, and is unidirectional. This increase is significant even when the phase difference between adjacent cilia is small. The obtained results provide guidelines for the optimal design of magnetically-driven artificial cilia for microfluidic propulsion.

Filter Feeding, Chaotic Filtration, and a Blinking Stokeslet

NASA Astrophysics Data System (ADS)

Blake, J. R.; Otto, S. R.; Blake, D. A.

The filtering mechanisms in bivalve molluscs, such as the mussel Mytilus edulis, and in sessile organisms, such as Vorticella or Stentor, involve complex fluid mechanical phenomena. In the former example, three different sets of cilia serving different functions are involved in the process whereas in the sessile organisms the flexibility and contractile nature of the stalk may play an important role in increasing the filtering efficiency of the organisms. In both cases, beating microscopic cilia are the ``engines'' driving the fluid motion, so the fluid mechanics will be dominated entirely by viscous forces. A fluid mechanical model is developed for the filtering mechanism in mussels that enables estimates to be made of the pressure drop through the gill filaments due to (i) latero-frontal filtering cilia, (ii) the lateral (pumping) cilia, and (iii) through the non-ciliated zone of the ventral end of the filament. The velocity profile across the filaments indicates that a backflow can occur in the centre of the channel leading to the formation of two ``standing'' eddies which may drive particles towards the mucus-laden short cilia, the third set of cilia. Filter feeding in the sessile organisms is modelled by a point force above a rigid boundary. The point force periodically changes its point of application according to a given protocol (a blinking stokeslet). The resulting fluid field is illustrated via Poincaré sections and particle dispersion-showing the potential for a much improved filtering efficiency. Returning to filter feeding in bivalve molluscs, this concept is extended to a pair of blinking stokeslets above a rigid boundary to give insight into possible mechanisms for movement of food particles onto the short mucus-bearing cilia. The appendix contains a Latin and English version of an ``Ode of Achievement'' in celebration of Sir James Lighthill's contributions to mathematics and fluid mechanics.

Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways.

PubMed

Sigg, Monika Abedin; Menchen, Tabea; Lee, Chanjae; Johnson, Jeffery; Jungnickel, Melissa K; Choksi, Semil P; Garcia, Galo; Busengdal, Henriette; Dougherty, Gerard W; Pennekamp, Petra; Werner, Claudius; Rentzsch, Fabian; Florman, Harvey M; Krogan, Nevan; Wallingford, John B; Omran, Heymut; Reiter, Jeremy F

2017-12-18

Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease. Copyright © 2017 Elsevier Inc. All rights reserved.

The NIMA-like kinase Nek2 is a key switch balancing cilia biogenesis and resorption in the development of left-right asymmetry.

PubMed

Endicott, S Joseph; Basu, Basudha; Khokha, Mustafa; Brueckner, Martina

2015-12-01

Vertebrate left-right (LR) asymmetry originates at a transient left-right organizer (LRO), a ciliated structure where cilia play a crucial role in breaking symmetry. However, much remains unknown about the choreography of cilia biogenesis and resorption at this organ. We recently identified a mutation affecting NEK2, a member of the NIMA-like serine-threonine kinase family, in a patient with congenital heart disease associated with abnormal LR development. Here, we report how Nek2 acts through cilia to influence LR patterning. Both overexpression and knockdown of nek2 in Xenopus result in abnormal LR development and reduction of LRO cilia count and motility, phenotypes that are modified by interaction with the Hippo signaling pathway. nek2 knockdown leads to a centriole defect at the LRO, consistent with the known role of Nek2 in centriole separation. Nek2 overexpression results in premature ciliary resorption in cultured cells dependent on function of the tubulin deacetylase Hdac6. Finally, we provide evidence that the known interaction between Nek2 and Nup98, a nucleoporin that localizes to the ciliary base, is important for regulating cilium resorption. Together, these data show that Nek2 is a switch balancing ciliogenesis and resorption in the development of LR asymmetry. © 2015. Published by The Company of Biologists Ltd.

Respiratory manifestations in 38 patients with Alström syndrome.

PubMed

Boerwinkle, Caroline; Marshall, Jan D; Bryant, Joy; Gahl, William A; Olivier, Kenneth N; Gunay-Aygun, Meral

2017-04-01

Alström syndrome (AS) is a rare, multi-system condition characterized by retinal degeneration, sensorineural hearing loss, obesity, insulin-resistant diabetes, hypertriglyceridemia, cardiomyopathy, hepatorenal disease, and recurrent respiratory infections. It belongs to a group of genetic disorders known as primary ciliopathies, which includes autosomal dominant and recessive polycystic kidney diseases, as well as Joubert and Bardet-Biedl syndromes. Prior studies have suggested phenotypic overlap between primary ciliopathies affecting the non-motile, sensory cilia, and primary ciliary dyskinesia (PCD), a motile ciliopathy characterized by respiratory tract disease. We describe the burden of oto-sino-pulmonary disease in 38 individuals with AS and examines the degree of clinical overlap between PCD and AS. Evaluation at the NIH Clinical Center included clinical examination, chest imaging, and clinical history surveys, as well as measurement of nasal nitric oxide (nNO) in nine patients. Recurrent otitis media was ubiquitous in the AS cohort (92%) with 50% requiring pressure equalization tube placement. A history of bronchitis/pneumonia and sinusitis was reported in 61% and 50% of individuals, respectively. PCD-characterizing symptoms (laterality defects, unexplained neonatal respiratory distress, year-round nasal congestion, and wet cough) were far less prevalent in the AS cohort compared to PCD, and the average nNO production in the AS cohort was 232 ± 57.1 nl/min compared to a cut-off of <77 nl/min for PCD. These data suggest that the oto-sino-respiratory complications in AS are prominent enough to warrant increased clinical attention, but significantly impaired respiratory cilia function as seen in PCD is unlikely in AS. (www.clinicaltrials.gov, trial NCT00068224) Pediatr Pulmonol. 2017;52:487-493. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Cycling of the signaling protein phospholipase D through cilia requires the BBSome only for the export phase

PubMed Central

Brown, Jason M.; Sampaio, Julio L.; Craft, Julie M.; Shevchenko, Andrej; Evans, James E.; Witman, George B.

2013-01-01

The BBSome is a complex of seven proteins, including BBS4, that is cycled through cilia by intraflagellar transport (IFT). Previous work has shown that the membrane-associated signaling protein phospholipase D (PLD) accumulates abnormally in cilia of Chlamydomonas reinhardtii bbs mutants. Here we show that PLD is a component of wild-type cilia but is enriched ∼150-fold in bbs4 cilia; this accumulation occurs progressively over time and results in altered ciliary lipid composition. When wild-type BBSomes were introduced into bbs cells, PLD was rapidly removed from the mutant cilia, indicating the presence of an efficient BBSome-dependent mechanism for exporting ciliary PLD. This export requires retrograde IFT. Importantly, entry of PLD into cilia is BBSome and IFT independent. Therefore, the BBSome is required only for the export phase of a process that continuously cycles PLD through cilia. Another protein, carbonic anhydrase 6, is initially imported normally into bbs4 cilia but lost with time, suggesting that its loss is a secondary effect of BBSome deficiency. PMID:23589493

Systematic VCP-UBXD Adaptor Network Proteomics Identifies a Role for UBXN10 in Regulating Ciliogenesis

PubMed Central

Raman, Malavika; Sergeev, Mikhail; Garnaas, Maija; Lydeard, John R.; Huttlin, Edward L.; Goessling, Wolfram; Shah, Jagesh V.; Harper, J. Wade

2015-01-01

The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to “segregate” ubiquitinated proteins from their binding partners. VCP acts via UBX-domain containing adaptors that provide target specificity, but targets and functions of UBXD proteins remain poorly understood. Through systematic proteomic analysis of UBXD proteins in human cells, we reveal a network of over 195 interacting proteins, implicating VCP in diverse cellular pathways. We have explored one such complex between an unstudied adaptor UBXN10 and the intraflagellar transport B (IFT-B) complex, which regulates anterograde transport into cilia. UBXN10 localizes to cilia in a VCP-dependent manner and both VCP and UBXN10 are required for ciliogenesis. Pharmacological inhibition of VCP destabilized the IFT-B complex and increased trafficking rates. Depletion of UBXN10 in zebrafish embryos causes defects in left-right asymmetry, which depends on functional cilia. This study provides a resource for exploring the landscape of UBXD proteins in biology and identifies an unexpected requirement for VCP-UBXN10 in ciliogenesis. PMID:26389662

Systematic proteomics of the VCP-UBXD adaptor network identifies a role for UBXN10 in regulating ciliogenesis.

PubMed

Raman, Malavika; Sergeev, Mikhail; Garnaas, Maija; Lydeard, John R; Huttlin, Edward L; Goessling, Wolfram; Shah, Jagesh V; Harper, J Wade

2015-10-01

The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to 'segregate' ubiquitylated proteins from their binding partners. VCP acts through UBX-domain-containing adaptors that provide target specificity, but the targets and functions of UBXD proteins remain poorly understood. Through systematic proteomic analysis of UBXD proteins in human cells, we reveal a network of over 195 interacting proteins, implicating VCP in diverse cellular pathways. We have explored one such complex between an unstudied adaptor UBXN10 and the intraflagellar transport B (IFT-B) complex, which regulates anterograde transport into cilia. UBXN10 localizes to cilia in a VCP-dependent manner and both VCP and UBXN10 are required for ciliogenesis. Pharmacological inhibition of VCP destabilized the IFT-B complex and increased trafficking rates. Depletion of UBXN10 in zebrafish embryos causes defects in left-right asymmetry, which depends on functional cilia. This study provides a resource for exploring the landscape of UBXD proteins in biology and identifies an unexpected requirement for VCP-UBXN10 in ciliogenesis.

Drosophila PLP assembles pericentriolar clouds that promote centriole stability, cohesion and MT nucleation

PubMed Central

Pratt, Metta B.; Johnson, Errin

2018-01-01

Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. It has been implicated in many aspects of centrosome and cilia function, but its precise role is unclear. Here, we examine Drosophila Pericentrin-like-protein (PLP) function in vivo in tissues that form both centrosomes and cilia. Plp mutant centrioles exhibit four major defects: (1) They are short and have subtle structural abnormalities; (2) They disengage prematurely, and so overduplicate; (3) They organise fewer cytoplasmic MTs during interphase; (4) When forming cilia, they fail to establish and/or maintain a proper connection to the plasma membrane—although, surprisingly, they can still form an axoneme-like structure that can recruit transition zone (TZ) proteins. We show that PLP helps assemble “pericentriolar clouds” of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. We propose that the partial loss of these structures may largely explain the complex centriole, centrosome and cilium defects we observe in Plp mutant cells. PMID:29425198

Forkhead Transcription Factor Fd3F Cooperates with Rfx to Regulate a Gene Expression Program for Mechanosensory Cilia Specialization

PubMed Central

Newton, Fay G.; zur Lage, Petra I.; Karak, Somdatta; Moore, Daniel J.; Göpfert, Martin C.; Jarman, Andrew P.

2012-01-01

Summary Cilia have evolved hugely diverse structures and functions to participate in a wide variety of developmental and physiological processes. Ciliary specialization requires differences in gene expression, but few transcription factors are known to regulate this, and their molecular function is unclear. Here, we show that the Drosophila Forkhead box (Fox) gene, fd3F, is required for specialization of the mechanosensory cilium of chordotonal (Ch) neurons. fd3F regulates genes for Ch-specific axonemal dyneins and TRPV ion channels, which are required for sensory transduction, and retrograde transport genes, which are required to differentiate their distinct motile and sensory ciliary zones. fd3F is reminiscent of vertebrate Foxj1, a motile cilia regulator, but fd3F regulates motility genes as part of a broader sensory regulation program. Fd3F cooperates with the pan-ciliary transcription factor, Rfx, to regulate its targets directly. This illuminates pathways involved in ciliary specialization and the molecular mechanism of transcription factors that regulate them. PMID:22698283

Constitutive Cyclin O deficiency results in penetrant hydrocephalus, impaired growth and infertility

PubMed Central

Núnez-Ollé, Marc; Jung, Carole; Terré, Berta; Balsiger, Norman A.; Plata, Cristina; Roset, Ramon; Pardo-Pastor, Carlos; Garrido, Marta; Rojas, Santiago; Alameda, Francesc; Lloreta, Josep; Martín-Caballero, Juan; Flores, Juana M.; Stracker, Travis H.; Valverde, Miguel A.; Muñoz, Francisco J.; Gil-Gómez, Gabriel

2017-01-01

Cyclin O (encoded by CCNO) is a member of the cyclin family with regulatory functions in ciliogenesis and apoptosis. Homozygous CCNO mutations have been identified in human patients with Reduced Generation of Multiple Motile Cilia (RGMC) and conditional inactivation of Ccno in the mouse recapitulates some of the pathologies associated with the human disease. These include defects in the development of motile cilia and hydrocephalus. To further investigate the functions of Ccno in vivo, we have generated a new mouse model characterized by the constitutive loss of Ccno in all tissues and followed a cohort during ageing. Ccno-/- mice were growth impaired and developed hydrocephalus with high penetrance. In addition, some Ccno+/- mice also developed hydrocephalus and affected Ccno-/- and Ccno+/- mice exhibited additional CNS defects including cortical thinning and hippocampal abnormalities. In addition to the CNS defects, both male and female Ccno-/- mice were infertile and female mice exhibited few motile cilia in the oviduct. Our results further establish CCNO as an important gene for normal development and suggest that heterozygous CCNO mutations could underlie hydrocephalus or diminished fertility in some human patients. PMID:29245899

Constitutive Cyclin O deficiency results in penetrant hydrocephalus, impaired growth and infertility.

PubMed

Núnez-Ollé, Marc; Jung, Carole; Terré, Berta; Balsiger, Norman A; Plata, Cristina; Roset, Ramon; Pardo-Pastor, Carlos; Garrido, Marta; Rojas, Santiago; Alameda, Francesc; Lloreta, Josep; Martín-Caballero, Juan; Flores, Juana M; Stracker, Travis H; Valverde, Miguel A; Muñoz, Francisco J; Gil-Gómez, Gabriel

2017-11-21

Cyclin O (encoded by CCNO ) is a member of the cyclin family with regulatory functions in ciliogenesis and apoptosis. Homozygous CCNO mutations have been identified in human patients with Reduced Generation of Multiple Motile Cilia (RGMC) and conditional inactivation of Ccno in the mouse recapitulates some of the pathologies associated with the human disease. These include defects in the development of motile cilia and hydrocephalus. To further investigate the functions of Ccno in vivo , we have generated a new mouse model characterized by the constitutive loss of Ccno in all tissues and followed a cohort during ageing. Ccno -/- mice were growth impaired and developed hydrocephalus with high penetrance. In addition, some Ccno +/- mice also developed hydrocephalus and affected Ccno -/- and Ccno +/- mice exhibited additional CNS defects including cortical thinning and hippocampal abnormalities. In addition to the CNS defects, both male and female Ccno -/- mice were infertile and female mice exhibited few motile cilia in the oviduct. Our results further establish CCNO as an important gene for normal development and suggest that heterozygous CCNO mutations could underlie hydrocephalus or diminished fertility in some human patients.

BLOC-1 is required for selective membrane protein trafficking from endosomes to primary cilia

PubMed Central

2017-01-01

Primary cilia perceive the extracellular environment through receptors localized in the ciliary membrane, but mechanisms directing specific proteins to this domain are poorly understood. To address this question, we knocked down proteins potentially important for ciliary membrane targeting and determined how this affects the ciliary trafficking of fibrocystin, polycystin-2, and smoothened. Our analysis showed that fibrocystin and polycystin-2 are dependent on IFT20, GMAP210, and the exocyst complex, while smoothened delivery is largely independent of these components. In addition, we found that polycystin-2, but not smoothened or fibrocystin, requires the biogenesis of lysosome-related organelles complex-1 (BLOC-1) for ciliary delivery. Consistent with the role of BLOC-1 in sorting from the endosome, we find that disrupting the recycling endosome reduces ciliary polycystin-2 and causes its accumulation in the recycling endosome. This is the first demonstration of a role for BLOC-1 in ciliary assembly and highlights the complexity of pathways taken to the cilium. PMID:28576874

An age of enlightenment for cilia: The FASEB Summer Research Conference on the “Biology of Cilia and Flagella”

PubMed Central

Tran, Pamela V.; Lechtreck, Karl F.

2015-01-01

From July 19–24, 2015, 169 clinicians and basic scientists gathered in the vertiginous heights of Snowmass, Colorado (2,502 m) for the fourth FASEB summer research conference on the ‘Biology of Cilia and Flagella’. Organizers Maureen Barr (Rutgers University), Iain Drummond (Massachusetts General Hospital/Harvard Medical School), and Jagesh Shah (Brigham and Women’s Hospital/Harvard Medical School) assembled a program filled with new data and forward-thinking ideas documenting the ongoing growth of the field. Sixty oral presentations and 77 posters covered novel aspects of cilia structure, ciliogenesis, cilia motility, cilia-mediated signaling, and cilia-related disease. In this report, we summarize the meeting, highlight exciting developments and discuss open questions. PMID:26597000

Centrioles initiate cilia assembly but are dispensable for maturation and maintenance in C. elegans.

PubMed

Serwas, Daniel; Su, Tiffany Y; Roessler, Max; Wang, Shaohe; Dammermann, Alexander

2017-06-05

Cilia are cellular projections that assemble on centriole-derived basal bodies. While cilia assembly is absolutely dependent on centrioles, it is not known to what extent they contribute to downstream events. The nematode C. elegans provides a unique opportunity to address this question, as centrioles do not persist at the base of mature cilia. Using fluorescence microscopy and electron tomography, we find that centrioles degenerate early during ciliogenesis. The transition zone and axoneme are not completely formed at this time, indicating that cilia maturation does not depend on intact centrioles. The hydrolethalus syndrome protein HYLS-1 is the only centriolar protein known to remain at the base of mature cilia and is required for intraflagellar transport trafficking. Surprisingly, targeted degradation of HYLS-1 after initiation of ciliogenesis does not affect ciliary structures. Taken together, our results indicate that while centrioles are essential to initiate cilia formation, they are dispensable for cilia maturation and maintenance. © 2017 Serwas et al.

Centrioles initiate cilia assembly but are dispensable for maturation and maintenance in C. elegans

PubMed Central

Roessler, Max

2017-01-01

Cilia are cellular projections that assemble on centriole-derived basal bodies. While cilia assembly is absolutely dependent on centrioles, it is not known to what extent they contribute to downstream events. The nematode C. elegans provides a unique opportunity to address this question, as centrioles do not persist at the base of mature cilia. Using fluorescence microscopy and electron tomography, we find that centrioles degenerate early during ciliogenesis. The transition zone and axoneme are not completely formed at this time, indicating that cilia maturation does not depend on intact centrioles. The hydrolethalus syndrome protein HYLS-1 is the only centriolar protein known to remain at the base of mature cilia and is required for intraflagellar transport trafficking. Surprisingly, targeted degradation of HYLS-1 after initiation of ciliogenesis does not affect ciliary structures. Taken together, our results indicate that while centrioles are essential to initiate cilia formation, they are dispensable for cilia maturation and maintenance. PMID:28411189

Emergence of multiple synchronization modes in hydrodynamically-coupled cilia

NASA Astrophysics Data System (ADS)

Guo, Hanliang; Kanso, Eva

2016-11-01

Motile cilia and flagella exhibit different phase coordinations. For example, closely swimming spermatozoa are observed to synchronize together; bi-flagellates Chlamydomonas regulate the flagella in a "breast-stroke" fashion; cilia on the surface of Paramecium beat in a fixed phase lag in an orchestrated wave like fashion. Experimental evidence suggests that phase coordinations can be achieved solely via hydrodynamical interactions. However, the exact mechanisms behind it remain illusive. Here, adapting a "geometric switch" model, we observe different synchronization modes in pairs of hydrodynamically-coupled cilia by changing physical parameters such as the strength of the cilia internal motor and the separation distance between cilia. Interestingly, we find regions in the parameter space where the coupled cilia reach stable phase coordinations and regions where the phase coordinations are sensitive to perturbations. We also find that leaning into the fluid reduces the sensitivity to perturbations, and produces stable phase coordination that is neither in-phase nor anti-phase, which could explain the origin of metachronal waves in large cilia populations.

Propulsion and trapping of microparticles by active cilia arrays.

PubMed

Bhattacharya, Amitabh; Buxton, Gavin A; Usta, O Berk; Balazs, Anna C

2012-02-14

We model the transport of a microscopic particle via a regular array of beating elastic cilia, whose tips experience an adhesive interaction with the particle's surface. At optimal adhesion strength, the average particle velocity is maximized. Using simulations spanning a range of cilia stiffness and cilia-particle adhesion strength, we explore the parameter space over which the particle can be "released", "propelled", or "trapped" by the cilia. We use a lower-order model to predict parameters for which the cilia are able to "propel" the particle. This is the first study that shows how both stiffness and adhesion strength are crucial for manipulation of particles by active cilia arrays. These results can facilitate the design of synthetic cilia that integrate adhesive and hydrodynamic interactions to selectively repel or trap particulates. Surfaces that are effective at repelling particulates are valuable for antifouling applications, while surfaces that can trap and, thus, remove particulates from the solution are useful for efficient filtration systems.

Notch/Her12 signalling modulates, motile/immotile cilia ratio downstream of Foxj1a in zebrafish left-right organizer

PubMed Central

Sampaio, Pedro; Pestana, Sara; Pinto, Andreia; Vaz, Andreia; Roxo-Rosa, Mónica; Gardner, Rui; Lopes, Telma; Schilling, Britta; Henry, Ian; Saúde, Leonor

2017-01-01

Foxj1a is necessary and sufficient to specify motile cilia. Using transcriptional studies and slow-scan two-photon live imaging capable of identifying the number of motile and immotile cilia, we now established that the final number of motile cilia depends on Notch signalling (NS). We found that despite all left-right organizer (LRO) cells express foxj1a and the ciliary axonemes of these cells have dynein arms, some cilia remain immotile. We identified that this decision is taken early in development in the Kupffer’s Vesicle (KV) precursors the readout being her12 transcription. We demonstrate that overexpression of either her12 or Notch intracellular domain (NICD) increases the number of immotile cilia at the expense of motile cilia, and leads to an accumulation of immotile cilia at the anterior half of the KV. This disrupts the normal fluid flow intensity and pattern, with consequent impact on dand5 expression pattern and left-right (L-R) axis establishment. PMID:28875937

WD60/FAP163 is a dynein intermediate chain required for retrograde intraflagellar transport in cilia

PubMed Central

Patel-King, Ramila S.; Gilberti, Renée M.; Hom, Erik F. Y.; King, Stephen M.

2013-01-01

Retrograde intraflagellar transport (IFT) is required for assembly of cilia. We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement. Biochemical analysis revealed that FAP163 is present in the flagellar matrix and is actively trafficked by IFT. Furthermore, FAP163 copurified with D1bIC(FAP133) and the LC8 dynein light chain, indicating that it is an integral component of the retrograde IFT dynein. To assess the functional role of FAP163, we generated an RNA interference knockdown of the orthologous protein (WD60) in planaria. The Smed-wd60(RNAi) animals had a severe ciliary assembly defect that dramatically compromised whole-organism motility. Most cilia were present as short stubs that had accumulated large quantities of IFT particle–like material between the doublet microtubules and the membrane. The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz. Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly. PMID:23864713

Primary ciliary dyskinesia.

PubMed

Lobo, L J; Zariwala, M A; Noone, P G

2014-09-01

Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder of cilia structure and function, leading to chronic infections of the respiratory tract, fertility problems and disorders of organ laterality. Making a definitive diagnosis is challenging, utilizing characteristic phenotypes, ciliary functional and ultra-structural defects in addition to newer screening tools such as nasal nitric oxide and genetic testing. There are 21 known PCD causing genes and in the future, comprehensive genetic testing may help diagnosis young infants prior to developing symptoms thus improving survival. Therapy includes surveillance of pulmonary function and microbiology in addition to, airway clearance, antibiotics and early referral to bronchiectasis centers. Standardized care at specialized centers using a multidisciplinary approach is likely to improve outcomes. In conjunction with the PCD foundation and lead investigators and clinicians are developing a network of PCD clinical centers to coordinate the effort in North America and Europe. As the network grows, care and knowledge will undoubtedly improve. © The Author 2014. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cilia are required for asymmetric nodal induction in the sea urchin embryo.

PubMed

Tisler, Matthias; Wetzel, Franziska; Mantino, Sabrina; Kremnyov, Stanislav; Thumberger, Thomas; Schweickert, Axel; Blum, Martin; Vick, Philipp

2016-08-23

Left-right (LR) organ asymmetries are a common feature of metazoan animals. In many cases, laterality is established by a conserved asymmetric Nodal signaling cascade during embryogenesis. In most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer (LRO), a ciliated epithelium present during gastrula/neurula stages. Conservation of LRO and flow beyond the vertebrates has not been reported yet. Here we study sea urchin embryos, which use nodal to establish larval LR asymmetry as well. Cilia were found in the archenteron of embryos undergoing gastrulation. Expression of foxj1 and dnah9 suggested that archenteron cilia were motile. Cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. High-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. Removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression. Our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos. Based on these results we argue that the anterior archenteron represents a bona fide LRO and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes.

A prefoldin-associated WD-repeat protein (WDR92) is required for the correct architectural assembly of motile cilia

PubMed Central

Patel-King, Ramila S.; King, Stephen M.

2016-01-01

WDR92 is a highly conserved WD-repeat protein that has been proposed to be involved in apoptosis and also to be part of a prefoldin-like cochaperone complex. We found that WDR92 has a phylogenetic signature that is generally compatible with it playing a role in the assembly or function of specifically motile cilia. To test this hypothesis, we performed an RNAi-based knockdown of WDR92 gene expression in the planarian Schmidtea mediterranea and were able to achieve a robust reduction in mRNA expression to levels undetectable under our standard RT-PCR conditions. We found that this treatment resulted in a dramatic reduction in the rate of organismal movement that was caused by a switch in the mode of locomotion from smooth, cilia-driven gliding to muscle-based, peristaltic contractions. Although the knockdown animals still assembled cilia of normal length and in similar numbers to controls, these structures had reduced beat frequency and did not maintain hydrodynamic coupling. By transmission electron microscopy we observed that many cilia had pleiomorphic defects in their architecture, including partial loss of dynein arms, incomplete closure of the B-tubule, and occlusion or replacement of the central pair complex by accumulated electron-dense material. These observations suggest that WDR92 is part of a previously unrecognized cytoplasmic chaperone system that is specifically required to fold key components necessary to build motile ciliary axonemes. PMID:26912790

Circular flow patterns induced by ciliary activity in reconstituted human bronchial epithelium

NASA Astrophysics Data System (ADS)

Viallat, Annie; Khelloufi, Kamel; Gras, Delphine; Chanez, Pascal; Aix Marseille Univ., CNRS, CINaM, Marseille, France Team; Aix Marseille Univ., CNRS, Inserm, LAI, Marseille, France Team

2016-11-01

Mucociliary clearance is the transport at the surface of airways of a complex fluid layer, the mucus, moved by the beats of microscopic cilia present on epithelial ciliated cells. We explored the coupling between the spatial organisation and the activity of cilia and the transport of surface fluids on reconstituted cultures of human bronchial epithelium at air-liquid interface, obtained by human biopsies. We reveal the existence of stable local circular surface flow patterns of mucus or Newtonian fluid at the epithelium surface. We find a power law over more than 3 orders of magnitude showing that the average ciliated cell density controls the size of these flow patterns, and, therefore the distance over which mucus can be transported. We show that these circular flow patterns result from the radial linear increase of the local propelling forces (due to ciliary beats) on each flow domain. This linear increase of local forces is induced by a fine self-regulation of both cilia density and orientation of ciliary beats. Local flow domains grow and merge during ciliogenesis to provide macroscopic mucus transport. This is possible only when the viscoelastic mucus continuously exerts a shear stress on beating cilia, revealing a mechanosensitive function of cilia. M. K. Khelloufi thanks the society MedBioMed for financial support. This work was supported by the ANR MUCOCIL project, Grant ANR-13-BSV5-0015 of the French Agence Nationale de la Recherche.

LRRC6 Mutation Causes Primary Ciliary Dyskinesia with Dynein Arm Defects

PubMed Central

Horani, Amjad; Ferkol, Thomas W.; Shoseyov, David; Wasserman, Mollie G.; Oren, Yifat S.; Kerem, Batsheva; Amirav, Israel; Cohen-Cymberknoh, Malena; Dutcher, Susan K.; Brody, Steven L.; Elpeleg, Orly; Kerem, Eitan

2013-01-01

Despite recent progress in defining the ciliome, the genetic basis for many cases of primary ciliary dyskinesia (PCD) remains elusive. We evaluated five children from two unrelated, consanguineous Palestinian families who had PCD with typical clinical features, reduced nasal nitric oxide concentrations, and absent dynein arms. Linkage analyses revealed a single common homozygous region on chromosome 8 and one candidate was conserved in organisms with motile cilia. Sequencing revealed a single novel mutation in LRRC6 (Leucine-rich repeat containing protein 6) that fit the model of autosomal recessive genetic transmission, leading to a change of a highly conserved amino acid from aspartic acid to histidine (Asp146His). LRRC6 was localized to the cytoplasm and was up-regulated during ciliogenesis in human airway epithelial cells in a Foxj1-dependent fashion. Nasal epithelial cells isolated from affected individuals and shRNA-mediated silencing in human airway epithelial cells, showed reduced LRRC6 expression, absent dynein arms, and slowed cilia beat frequency. Dynein arm proteins were either absent or mislocalized to the cytoplasm in airway epithelial cells from a primary ciliary dyskinesia subject. These findings suggest that LRRC6 plays a role in dynein arm assembly or trafficking and when mutated leads to primary ciliary dyskinesia with laterality defects. PMID:23527195

Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles.

PubMed

Kong, Dong; Farmer, Veronica; Shukla, Anil; James, Jana; Gruskin, Richard; Kiriyama, Shigeo; Loncarek, Jadranka

2014-09-29

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.

Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles

PubMed Central

Kong, Dong; Farmer, Veronica; Shukla, Anil; James, Jana; Gruskin, Richard; Kiriyama, Shigeo

2014-01-01

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells. PMID:25246616

Subnanometre-resolution structure of the doublet microtubule reveals new classes of microtubule-associated proteins

PubMed Central

Ichikawa, Muneyoshi; Liu, Dinan; Kastritis, Panagiotis L.; Basu, Kaustuv; Hsu, Tzu Chin; Yang, Shunkai; Bui, Khanh Huy

2017-01-01

Cilia are ubiquitous, hair-like appendages found in eukaryotic cells that carry out functions of cell motility and sensory reception. Cilia contain an intriguing cytoskeletal structure, termed the axoneme that consists of nine doublet microtubules radially interlinked and longitudinally organized in multiple specific repeat units. Little is known, however, about how the axoneme allows cilia to be both actively bendable and sturdy or how it is assembled. To answer these questions, we used cryo-electron microscopy to structurally analyse several of the repeating units of the doublet at sub-nanometre resolution. This structural detail enables us to unambiguously assign α- and β-tubulins in the doublet microtubule lattice. Our study demonstrates the existence of an inner sheath composed of different kinds of microtubule inner proteins inside the doublet that likely stabilizes the structure and facilitates the specific building of the B-tubule. PMID:28462916

Acoustic actuation of in situ fabricated artificial cilia

NASA Astrophysics Data System (ADS)

Orbay, Sinem; Ozcelik, Adem; Bachman, Hunter; Huang, Tony Jun

2018-02-01

We present on-chip acoustic actuation of in situ fabricated artificial cilia. Arrays of cilia structures are UV polymerized inside a microfluidic channel using a photocurable polyethylene glycol (PEG) polymer solution and photomasks. During polymerization, cilia structures are attached to a silane treated glass surface inside the microchannel. Then, the cilia structures are actuated using acoustic vibrations at 4.6 kHz generated by piezo transducers. As a demonstration of a practical application, DI water and fluorescein dye solutions are mixed inside a microfluidic channel. Using pulses of acoustic excitations, and locally fabricated cilia structures within a certain region of the microchannel, a waveform of mixing behavior is obtained. This result illustrates one potential application wherein researchers can achieve spatiotemporal control of biological microenvironments in cell stimulation studies. These acoustically actuated, in situ fabricated, cilia structures can be used in many on-chip applications in biological, chemical and engineering studies.

The Developmental Process of the Growing Motile Ciliary Tip Region.

PubMed

Reynolds, Matthew J; Phetruen, Tanaporn; Fisher, Rebecca L; Chen, Ke; Pentecost, Brian T; Gomez, George; Ounjai, Puey; Sui, Haixin

2018-05-22

Eukaryotic motile cilia/flagella play vital roles in various physiological processes in mammals and some protists. Defects in cilia formation underlie multiple human disorders, known as ciliopathies. The detailed processes of cilia growth and development are still far from clear despite extensive studies. In this study, we characterized the process of cilium formation (ciliogenesis) by investigating the newly developed motile cilia of deciliated protists using complementary techniques in electron microscopy and image analysis. Our results demonstrated that the distal tip region of motile cilia exhibit progressive morphological changes as cilia develop. This developmental process is time-dependent and continues after growing cilia reach their full lengths. The structural analysis of growing ciliary tips revealed that B-tubules of axonemal microtubule doublets terminate far away from the tip end, which is led by the flagellar tip complex (FTC), demonstrating that the FTC might not directly mediate the fast turnover of intraflagellar transport (IFT).

Defective ciliogenesis, embryonic lethality and severe impairment of the Sonic Hedgehog pathway caused by inactivation of the mouse complex A intraflagellar transport gene Ift122/Wdr10, partially overlapping with the DNA repair gene Med1/Mbd4

PubMed Central

Cortellino, Salvatore; Wang, Chengbing; Wang, Baolin; Bassi, Maria Rosaria; Caretti, Elena; Champeval, Delphine; Calmont, Amelie; Jarnik, Michal; Burch, John; Zaret, Kenneth; Larue, Lionel; Bellacosa, Alfonso

2009-01-01

Primary cilia are assembled and maintained by evolutionarily conserved intraflagellar transport (IFT) proteins that are involved in the coordinated movement of macromolecular cargo from the basal body to the cilium tip and back. The IFT machinery is organized in two structural complexes named complex A and complex B. Recently, inactivation in the mouse germline of Ift genes belonging to complex B revealed a requirement of ciliogenesis, or proteins involved in ciliogenesis, for Sonic Hedgehog (Shh) signaling in mammals. Here we report on a complex A mutant mouse, defective for the Ift122 gene. Ift122-null embryos show multiple developmental defects (exencephaly, situs viscerum inversus, delay in turning, hemorrhage and defects in limb development) that result in lethality. In the node, primary cilia were absent or malformed in homozygous mutant and heterozygous embryos, respectively. Impairment of the Shh pathway was apparent in both neural tube patterning (expansion of motoneurons and rostro-caudal level-dependent contraction or expansion of the dorso-lateral interneurons), and limb patterning (ectrosyndactyly). These phenotypes are distinct from both complex B IFT mutant embryos and embryos defective for the ciliary protein hennin/Arl13b, and suggest reduced levels of both Gli2/Gli3 activator and Gli3 repressor functions. We conclude that complex A and complex B factors play similar but distinct roles in ciliogenesis and Shh/Gli3 signaling. PMID:19000668

Microfluidic active mixers employing ultra-high aspect-ratio rare-earth magnetic nano-composite polymer artificial cilia

NASA Astrophysics Data System (ADS)

Rahbar, Mona; Shannon, Lesley; Gray, Bonnie L.

2014-02-01

We present a new micromixer based on highly magnetic, flexible, high aspect-ratio, artificial cilia that are fabricated as individual micromixer elements or in arrays for improved mixing performance. These new cilia enable high efficiency, fast mixing in a microchamber, and are controlled by small electromagnetic fields. The artificial cilia are fabricated using a new micromolding process for nano-composite polymers. Cilia fibers with aspect-ratios as high as 8:0.13 demonstrate the fabrication technique's capability in creating ultra-high aspect-ratio microstructures. Cilia, which are realized in polydimethylsiloxane doped with rare-earth magnetic powder, are magnetized to produce permanent magnetic structures with bidirectional deflection capabilities, making them highly suitable as mixers controlled by electromagnetic fields. Due to the high magnetization level of the polarized nano-composite polymer, we are able to use miniature electromagnets providing relatively small magnetic fields of 1.1 to 7 mT to actuate the cilia microstructures over a very wide motion range. Mixing performances of a single cilium, as well as different arrays of multiple cilia ranging from 2 to 8 per reaction chamber, are characterized and compared with passive diffusion mixing performance. The mixer cilia are actuated at different amplitudes and frequencies to optimize mixing performance. We demonstrate that more than 85% of the total volume of the reaction chamber is fully mixed after 3.5 min using a single cilium mixer at 7 mT compared with only 20% of the total volume mixed with passive diffusion. The time to achieve over 85% mixing is further reduced to 70 s using an array of eight cilia microstructures. The novel microfabrication technique and use of rare-earth permanently-magnetizable nano-composite polymers in mixer applications has not been reported elsewhere by other researchers. We further demonstrate improved mixing over other cilia micromixers as enabled by the high aspect-ratio, high flexibility, and magnetic properties of our cilia micromixer elements.

Seeing cilia: imaging modalities for ciliary motion and clinical connections.

PubMed

Peabody, Jacelyn E; Shei, Ren-Jay; Bermingham, Brent M; Phillips, Scott E; Turner, Brett; Rowe, Steven M; Solomon, George M

2018-06-01

The respiratory tract is lined with multiciliated epithelial cells that function to move mucus and trapped particles via the mucociliary transport apparatus. Genetic and acquired ciliopathies result in diminished mucociliary clearance, contributing to disease pathogenesis. Recent innovations in imaging technology have advanced our understanding of ciliary motion in health and disease states. Application of imaging modalities including transmission electron microscopy, high-speed video microscopy, and micron-optical coherence tomography could improve diagnostics and be applied for precision medicine. In this review, we provide an overview of ciliary motion, imaging modalities, and ciliopathic diseases of the respiratory system including primary ciliary dyskinesia, cystic fibrosis, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis.

Swimming like algae: biomimetic soft artificial cilia

PubMed Central

Sareh, Sina; Rossiter, Jonathan; Conn, Andrew; Drescher, Knut; Goldstein, Raymond E.

2013-01-01

Cilia are used effectively in a wide variety of biological systems from fluid transport to thrust generation. Here, we present the design and implementation of artificial cilia, based on a biomimetic planar actuator using soft-smart materials. This actuator is modelled on the cilia movement of the alga Volvox, and represents the cilium as a piecewise constant-curvature robotic actuator that enables the subsequent direct translation of natural articulation into a multi-segment ionic polymer metal composite actuator. It is demonstrated how the combination of optimal segmentation pattern and biologically derived per-segment driving signals reproduce natural ciliary motion. The amenability of the artificial cilia to scaling is also demonstrated through the comparison of the Reynolds number achieved with that of natural cilia. PMID:23097503

Microfabrication of IPMC cilia for bio-inspired flow sensing

NASA Astrophysics Data System (ADS)

Lei, Hong; Li, Wen; Tan, Xiaobo

2012-04-01

As the primary flow sensing organ for fishes, the lateral line system plays a critical role in fish behavior. Analogous to its biological counterpart, an artificial lateral line system, consisting of arrays of micro flow sensors, is expected to be instrumental in the navigation and control of underwater robots. In this paper we investigate the microfabrication of ionic polymer-metal composite (IPMC) cilia for the purpose of flow sensing. While existing macro- and microfabrication methods for IPMCs have predominantly focused on planar structures, we propose a device where micro IPMC beams stand upright on a substrate to effectively interact with the flow. Challenges in the casting of 3D Nafion structure and selective formation of electrodes are discussed, and potential solutions for addressing these challenges are presented together with preliminary microfabrication results.

Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation.

PubMed

Vladar, Eszter K; Nayak, Jayakar V; Milla, Carlos E; Axelrod, Jeffrey D

2016-08-18

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.

Mechanism for generation of left isomerism in Ccdc40 mutant embryos

PubMed Central

Sugrue, Kelsey F.

2017-01-01

Leftward fluid flow in the mouse node is generated by cilia and is critical for initiating asymmetry of the left-right axis. Coiled-coil domain containing-40 (Ccdc40) plays an evolutionarily conserved role in the assembly of motile cilia and establishment of the left-right axis. Approximately one-third of Ccdc40lnks mutant embryos display situs defects and here we investigate the underlying mechanism. Ccdc40lnks mutants show delayed induction of markers of the left-lateral plate mesoderm (L-LPM) including Lefty1, Lefty2 and Nodal. Consistent with defective cilia motility compromising fluid flow across the node, initiation of asymmetric perinodal Cerberus like-2 (Cerl2) expression is delayed and then randomized. This is followed by delayed and then randomized asymmetric Nodal expression around the node. We propose a model to explain how left isomerism arises in a proportion of Ccdc40lnks mutants. We postulate that with defective motile cilia, Cerl2 expression remains symmetric and Nodal is antagonized equally on both sides of the node. This effectively reduces Nodal activation bilaterally, leading to reduced and delayed activation of Nodal and its antagonists in the LPM. This model is further supported by the failure to establish Nodal expression in the left-LPM with reduced Nodal gene dosage in Ccdc40lnks/lnks;NodalLacZ/+ mutants causing a predominance of right not left isomerism. Together these results suggest a model where cilia generated fluid flow in the node functions to ensure robust Nodal activation and a timely left-sided developmental program in the LPM. PMID:28182636

Somatostatin Signaling in Neuronal Cilia Is Criticalfor Object Recognition Memory

PubMed Central

Einstein, Emily B.; Patterson, Carlyn A.; Hon, Beverly J.; Regan, Kathleen A.; Reddi, Jyoti; Melnikoff, David E.; Mateer, Marcus J.; Schulz, Stefan; Johnson, Brian N.

2010-01-01

Most neurons possess a single, nonmotile cilium that projects out from the cell surface. These microtubule-based organelles are important in brain development and neurogenesis; however, their function in mature neurons is unknown. Cilia express a complement of proteins distinct from other neuronal compartments, one of which is the somatostatin receptor subtype SST3. We show here that SST3 is critical for object recognition memory in mice. sst3 knock-out mice are severely impaired in discriminating novel objects, whereas they retain normal memory for object location. Further, systemic injection of an SST3 antagonist (ACQ090) disrupts recall of familiar objects in wild-type mice. To examine mechanisms of SST3, we tested synaptic plasticity in CA1 hippocampus. Electrically evoked long-term potentiation (LTP) was normal in sst3 knock-out mice, while adenylyl cyclase/cAMP-mediated LTP was impaired. The SST3 antagonist also disrupted cAMP-mediated LTP. Basal cAMP levels in hippocampal lysate were reduced in sst3 knock-out mice compared with wild-type mice, while the forskolin-induced increase in cAMP levels was normal. The SST3 antagonist inhibited forskolin-stimulated cAMP increases, whereas the SST3 agonist L-796,778 increased basal cAMP levels in hippocampal slices but not hippocampal lysate. Our results show that somatostatin signaling in neuronal cilia is critical for recognition memory and suggest that the cAMP pathway is a conserved signaling motif in cilia. Neuronal cilia therefore represent a novel nonsynaptic compartment crucial for signaling involved in a specific form of synaptic plasticity and in novelty detection. PMID:20335466

Methods for imaging individual cilia in living echinoid embryos.

PubMed

Morris, Robert L; Pope, Hans W; Sholi, Adam N; Williams, Leah M; Ettinger, Chelsea R; Beacham, Gwendolyn M; Shintaku, Tatsushi; Abbott, Zachary D; Doherty, Elyse M

2015-01-01

The embryos of echinoids (sea urchins and sand dollars) serve as excellent models for studying cilia differentiation and stages of the cilia life cycle including ciliogenic initiation, growth, maintenance, and retraction. Early in echinoid development, uniform motile cilia form on all cells simultaneously but then rapidly differentiate into multiple cilia types that differ in morphology, motility, and signaling sensitivity. Metal ion treatments that shift germ layer boundaries and thereby "animalize" or "vegetalize" embryos can be used to enrich for low-abundance cilia types rendering those specialized cilia and the differentiation processes they exhibit much easier to study. The experimental advantages of having robust cilia growth and differentiation is tempered by the challenge of restraining ciliated embryos well enough to view the process of ciliogenesis live. We have developed four observation chambers as modifications of the Kiehart chamber for long-term light microscopic imaging of ciliated echinoid embryos. One of these systems employs paramagnetic beads to render ciliated larvae magnetic so they can be gently and reversibly trapped directly under the objective lens. With this magnetic trapping system, the larva can be positioned and repositioned until they achieve the orientation with the clearest view of any cilia of interest. These methods of gentle embryo restraint allow normal embryo development and the normal ciliogenic cycle and ciliary differentiation processes to continue in direct view. Sequential image series can then be collected and analyzed to quantitatively study the wide spectrum of cilia behaviors and properties that arise in developing echinoid embryos. Copyright © 2015. Published by Elsevier Inc.

Particle sorting by Paramecium cilia arrays.

PubMed

Mayne, Richard; Whiting, James G H; Wheway, Gabrielle; Melhuish, Chris; Adamatzky, Andrew

Motile cilia are cell-surface organelles whose purposes, in ciliated protists and certain ciliated metazoan epithelia, include generating fluid flow, sensing and substance uptake. Certain properties of cilia arrays, such as beating synchronisation and manipulation of external proximate particulate matter, are considered emergent, but remain incompletely characterised despite these phenomena having being the subject of extensive modelling. This study constitutes a laboratory experimental characterisation of one of the emergent properties of motile cilia: manipulation of adjacent particulates. The work demonstrates through automated videomicrographic particle tracking that interactions between microparticles and somatic cilia arrays of the ciliated model organism Paramecium caudatum constitute a form of rudimentary 'sorting'. Small particles are drawn into the organism's proximity by cilia-induced fluid currents at all times, whereas larger particles may be held immobile at a distance from the cell margin when the cell generates characteristic feeding currents in the surrounding media. These findings can contribute to the design and fabrication of biomimetic cilia, with potential applications to the study of ciliopathies. Copyright © 2017 Elsevier B.V. All rights reserved.

Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival.

PubMed

Omori, Yoshihiro; Chaya, Taro; Katoh, Kimiko; Kajimura, Naoko; Sato, Shigeru; Muraoka, Koichiro; Ueno, Shinji; Koyasu, Toshiyuki; Kondo, Mineo; Furukawa, Takahisa

2010-12-28

Cilia function as cell sensors in many organs, and their disorders are referred to as "ciliopathies." Although ciliary components and transport machinery have been well studied, regulatory mechanisms of ciliary formation and maintenance are poorly understood. Here we show that male germ cell-associated kinase (Mak) regulates retinal photoreceptor ciliary length and subcompartmentalization. Mak was localized both in the connecting cilia and outer-segment axonemes of photoreceptor cells. In the Mak-null retina, photoreceptors exhibit elongated cilia and progressive degeneration. We observed accumulation of intraflagellar transport 88 (IFT88) and IFT57, expansion of kinesin family member 3A (Kif3a), and acetylated α-tubulin signals in the Mak-null photoreceptor cilia. We found abnormal rhodopsin accumulation in the Mak-null photoreceptor cell bodies at postnatal day 14. In addition, overexpression of retinitis pigmentosa 1 (RP1), a microtubule-associated protein localized in outer-segment axonemes, induced ciliary elongation, and Mak coexpression rescued excessive ciliary elongation by RP1. The RP1 N-terminal portion induces ciliary elongation and increased intensity of acetylated α-tubulin labeling in the cells and is phosphorylated by Mak. These results suggest that Mak is essential for the regulation of ciliary length and is required for the long-term survival of photoreceptors.

Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival

PubMed Central

Omori, Yoshihiro; Chaya, Taro; Katoh, Kimiko; Kajimura, Naoko; Sato, Shigeru; Muraoka, Koichiro; Ueno, Shinji; Koyasu, Toshiyuki; Kondo, Mineo; Furukawa, Takahisa

2010-01-01

Cilia function as cell sensors in many organs, and their disorders are referred to as “ciliopathies.” Although ciliary components and transport machinery have been well studied, regulatory mechanisms of ciliary formation and maintenance are poorly understood. Here we show that male germ cell-associated kinase (Mak) regulates retinal photoreceptor ciliary length and subcompartmentalization. Mak was localized both in the connecting cilia and outer-segment axonemes of photoreceptor cells. In the Mak-null retina, photoreceptors exhibit elongated cilia and progressive degeneration. We observed accumulation of intraflagellar transport 88 (IFT88) and IFT57, expansion of kinesin family member 3A (Kif3a), and acetylated α-tubulin signals in the Mak-null photoreceptor cilia. We found abnormal rhodopsin accumulation in the Mak-null photoreceptor cell bodies at postnatal day 14. In addition, overexpression of retinitis pigmentosa 1 (RP1), a microtubule-associated protein localized in outer-segment axonemes, induced ciliary elongation, and Mak coexpression rescued excessive ciliary elongation by RP1. The RP1 N-terminal portion induces ciliary elongation and increased intensity of acetylated α-tubulin labeling in the cells and is phosphorylated by Mak. These results suggest that Mak is essential for the regulation of ciliary length and is required for the long-term survival of photoreceptors. PMID:21148103

Do Deregulated Cas Proteins Induce Genomic Instability In Early Stage Ovarian Cancer?

DTIC Science & Technology

2007-12-01

3457–3467. Ezratty, E.J., Partridge, M.A., and Gundersen, G.G. (2005). Microtu- bule -induced focal adhesion disassembly is mediated by dynamin and... bule -associated deacetylase. Nature 417, 455–458. Iomini, C., Tejada, K., Mo, W., Vaananen, H., and Piperno, G. (2004). Primary cilia of human

Loss of Nephrocystin-3 Function Can Cause Embryonic Lethality, Meckel-Gruber-like Syndrome, Situs Inversus, and Renal-Hepatic-Pancreatic Dysplasia

PubMed Central

Bergmann, Carsten; Fliegauf, Manfred; Brüchle, Nadina Ortiz; Frank, Valeska; Olbrich, Heike; Kirschner, Jan; Schermer, Bernhard; Schmedding, Ingolf; Kispert, Andreas; Kränzlin, Bettina; Nürnberg, Gudrun; Becker, Christian; Grimm, Tiemo; Girschick, Gundula; Lynch, Sally A.; Kelehan, Peter; Senderek, Jan; Neuhaus, Thomas J.; Stallmach, Thomas; Zentgraf, Hanswalter; Nürnberg, Peter; Gretz, Norbert; Lo, Cecilia; Lienkamp, Soeren; Schäfer, Tobias; Walz, Gerd; Benzing, Thomas; Zerres, Klaus; Omran, Heymut

2008-01-01

Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling. PMID:18371931

Acentriolar mitosis activates a p53-dependent apoptosis pathway in the mouse embryo

PubMed Central

Bazzi, Hisham; Anderson, Kathryn V.

2014-01-01

Centrosomes are the microtubule-organizing centers of animal cells that organize interphase microtubules and mitotic spindles. Centrioles are the microtubule-based structures that organize centrosomes, and a defined set of proteins, including spindle assembly defective-4 (SAS4) (CPAP/CENPJ), is required for centriole biogenesis. The biological functions of centrioles and centrosomes vary among animals, and the functions of mammalian centrosomes have not been genetically defined. Here we use a null mutation in mouse Sas4 to define the cellular and developmental functions of mammalian centrioles in vivo. Sas4-null embryos lack centrosomes but survive until midgestation. As expected, Sas4−/− mutants lack primary cilia and therefore cannot respond to Hedgehog signals, but other developmental signaling pathways are normal in the mutants. Unlike mutants that lack cilia, Sas4−/− embryos show widespread apoptosis associated with global elevated expression of p53. Cell death is rescued in Sas4−/− p53−/− double-mutant embryos, demonstrating that mammalian centrioles prevent activation of a p53-dependent apoptotic pathway. Expression of p53 is not activated by abnormalities in bipolar spindle organization, chromosome segregation, cell-cycle profile, or DNA damage response, which are normal in Sas4−/− mutants. Instead, live imaging shows that the duration of prometaphase is prolonged in the mutants while two acentriolar spindle poles are assembled. Independent experiments show that prolonging spindle assembly is sufficient to trigger p53-dependent apoptosis. We conclude that a short delay in the prometaphase caused by the absence of centrioles activates a previously undescribed p53-dependent cell death pathway in the rapidly dividing cells of the mouse embryo. PMID:24706806

Loss of FTO antagonises Wnt signaling and leads to developmental defects associated with ciliopathies.

PubMed

Osborn, Daniel P S; Roccasecca, Rosa Maria; McMurray, Fiona; Hernandez-Hernandez, Victor; Mukherjee, Sriparna; Barroso, Inês; Stemple, Derek; Cox, Roger; Beales, Philip L; Christou-Savina, Sonia

2014-01-01

Common intronic variants in the Human fat mass and obesity-associated gene (FTO) are found to be associated with an increased risk of obesity. Overexpression of FTO correlates with increased food intake and obesity, whilst loss-of-function results in lethality and severe developmental defects. Despite intense scientific discussions around the role of FTO in energy metabolism, the function of FTO during development remains undefined. Here, we show that loss of Fto leads to developmental defects such as growth retardation, craniofacial dysmorphism and aberrant neural crest cells migration in Zebrafish. We find that the important developmental pathway, Wnt, is compromised in the absence of FTO, both in vivo (zebrafish) and in vitro (Fto(-/-) MEFs and HEK293T). Canonical Wnt signalling is down regulated by abrogated β-Catenin translocation to the nucleus whilst non-canonical Wnt/Ca(2+) pathway is activated via its key signal mediators CaMKII and PKCδ. Moreover, we demonstrate that loss of Fto results in short, absent or disorganised cilia leading to situs inversus, renal cystogenesis, neural crest cell defects and microcephaly in Zebrafish. Congruently, Fto knockout mice display aberrant tissue specific cilia. These data identify FTO as a protein-regulator of the balanced activation between canonical and non-canonical branches of the Wnt pathway. Furthermore, we present the first evidence that FTO plays a role in development and cilia formation/function.

Breakup and then makeup: a predictive model of how cilia self-regulate hardness for posture control.

PubMed

Bandyopadhyay, Promode R; Hansen, Joshua C

2013-01-01

Functioning as sensors and propulsors, cilia are evolutionarily conserved organelles having a highly organized internal structure. How a paramecium's cilium produces off-propulsion-plane curvature during its return stroke for symmetry breaking and drag reduction is not known. We explain these cilium deformations by developing a torsional pendulum model of beat frequency dependence on viscosity and an olivo-cerebellar model of self-regulation of posture control. The phase dependence of cilia torsion is determined, and a bio-physical model of hardness control with predictive features is offered. Crossbridge links between the central microtubule pair harden the cilium during the power stroke; this stroke's end is a critical phase during which ATP molecules soften the crossbridge-microtubule attachment at the cilium inflection point where torsion is at its maximum. A precipitous reduction in hardness ensues, signaling the start of ATP hydrolysis that re-hardens the cilium. The cilium attractor basin could be used as reference for perturbation sensing.

Breakup and then makeup: a predictive model of how cilia self-regulate hardness for posture control

NASA Astrophysics Data System (ADS)

Bandyopadhyay, Promode R.; Hansen, Joshua C.

2013-06-01

Functioning as sensors and propulsors, cilia are evolutionarily conserved organelles having a highly organized internal structure. How a paramecium's cilium produces off-propulsion-plane curvature during its return stroke for symmetry breaking and drag reduction is not known. We explain these cilium deformations by developing a torsional pendulum model of beat frequency dependence on viscosity and an olivo-cerebellar model of self-regulation of posture control. The phase dependence of cilia torsion is determined, and a bio-physical model of hardness control with predictive features is offered. Crossbridge links between the central microtubule pair harden the cilium during the power stroke; this stroke's end is a critical phase during which ATP molecules soften the crossbridge-microtubule attachment at the cilium inflection point where torsion is at its maximum. A precipitous reduction in hardness ensues, signaling the start of ATP hydrolysis that re-hardens the cilium. The cilium attractor basin could be used as reference for perturbation sensing.

Breakup and then makeup: a predictive model of how cilia self-regulate hardness for posture control

PubMed Central

Bandyopadhyay, Promode R.; Hansen, Joshua C.

2013-01-01

Functioning as sensors and propulsors, cilia are evolutionarily conserved organelles having a highly organized internal structure. How a paramecium's cilium produces off-propulsion-plane curvature during its return stroke for symmetry breaking and drag reduction is not known. We explain these cilium deformations by developing a torsional pendulum model of beat frequency dependence on viscosity and an olivo-cerebellar model of self-regulation of posture control. The phase dependence of cilia torsion is determined, and a bio-physical model of hardness control with predictive features is offered. Crossbridge links between the central microtubule pair harden the cilium during the power stroke; this stroke's end is a critical phase during which ATP molecules soften the crossbridge-microtubule attachment at the cilium inflection point where torsion is at its maximum. A precipitous reduction in hardness ensues, signaling the start of ATP hydrolysis that re-hardens the cilium. The cilium attractor basin could be used as reference for perturbation sensing. PMID:23739771

Discrete multi-physics simulations of diffusive and convective mass transfer in boundary layers containing motile cilia in lungs.

PubMed

Ariane, Mostapha; Kassinos, Stavros; Velaga, Sitaram; Alexiadis, Alessio

2018-04-01

In this paper, the mass transfer coefficient (permeability) of boundary layers containing motile cilia is investigated by means of discrete multi-physics. The idea is to understand the main mechanisms of mass transport occurring in a ciliated-layer; one specific application being inhaled drugs in the respiratory epithelium. The effect of drug diffusivity, cilia beat frequency and cilia flexibility is studied. Our results show the existence of three mass transfer regimes. A low frequency regime, which we called shielding regime, where the presence of the cilia hinders mass transport; an intermediate frequency regime, which we have called diffusive regime, where diffusion is the controlling mechanism; and a high frequency regime, which we have called convective regime, where the degree of bending of the cilia seems to be the most important factor controlling mass transfer in the ciliated-layer. Since the flexibility of the cilia and the frequency of the beat changes with age and health conditions, the knowledge of these three regimes allows prediction of how mass transfer varies with these factors. Copyright © 2018 Elsevier Ltd. All rights reserved.

Asynchronous beating of cilia enhances particle capture rate

NASA Astrophysics Data System (ADS)

Ding, Yang; Kanso, Eva

2014-11-01

Many aquatic micro-organisms use beating cilia to generate feeding currents and capture particles in surrounding fluids. One of the capture strategies is to ``catch up'' with particles when a cilium is beating towards the overall flow direction (effective stroke) and intercept particles on the downstream side of the cilium. Here, we developed a 3D computational model of a cilia band with prescribed motion in a viscous fluid and calculated the trajectories of the particles with different sizes in the fluid. We found an optimal particle diameter that maximizes the capture rate. The flow field and particle motion indicate that the low capture rate of smaller particles is due to the laminar flow in the neighbor of the cilia, whereas larger particles have to move above the cilia tips to get advected downstream which decreases their capture rate. We then analyzed the effect of beating coordination between neighboring cilia on the capture rate. Interestingly, we found that asynchrony of the beating of the cilia can enhance the relative motion between a cilium and the particles near it and hence increase the capture rate.

Primary cilia in gastric Gastrointestinal Stromal Tumours (GISTs): an ultrastructural study

PubMed Central

Castiella, Tomás; Muñoz, Guillermo; Luesma, María José; Santander, Sonia; Soriano, Mario; Junquera, Concepción

2013-01-01

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal (non-epithelial) neoplasms of the human gastrointestinal (GI) tract. They are thought to derive from interstitial cells of Cajal (ICCs) or an ICC progenitor based on immunophenotypical and ultrastructural similarities. Because ICCs show primary cilium, our hypothesis is based on the possibility that some of these neoplastic cells could also present it. To determine this, an exhaustive ultrastructural study has been developed on four gastric GISTs. Previous studies had demonstrated considerable variability in tumour cells with two dominating phenotypes, spindly and epithelioid. In addition to these two types, we have found another cell type reminiscent of adult ICCs with a voluminous nucleus surrounded by narrow perinuclear cytoplasm with long slender cytoplasmic processes. We have also noted the presence of small undifferentiated cells. In this study, we report for the first time the presence of primary cilia (PCs) in spindle and epithelioid tumour cells, an ultrastructural feature we consider of special interest that has hitherto been ignored in the literature dealing with the ultrastructure of GISTs. We also point out the frequent occurrence of multivesicular bodies (MVBs). The ultrastructural findings described in gastric GISTs in this study appear to be relevant considering the critical roles played by PCs and MVBs recently demonstrated in tumourigenic processes. PMID:23672577

Loss of ift122, a Retrograde Intraflagellar Transport (IFT) Complex Component, Leads to Slow, Progressive Photoreceptor Degeneration Due to Inefficient Opsin Transport.

PubMed

Boubakri, Meriam; Chaya, Taro; Hirata, Hiromi; Kajimura, Naoko; Kuwahara, Ryusuke; Ueno, Akiko; Malicki, Jarema; Furukawa, Takahisa; Omori, Yoshihiro

2016-11-18

In the retina, aberrant opsin transport from cell bodies to outer segments leads to retinal degenerative diseases such as retinitis pigmentosa. Opsin transport is facilitated by the intraflagellar transport (IFT) system that mediates the bidirectional movement of proteins within cilia. In contrast to functions of the anterograde transport executed by IFT complex B (IFT-B), the precise functions of the retrograde transport mediated by IFT complex A (IFT-A) have not been well studied in photoreceptor cilia. Here, we analyzed developing zebrafish larvae carrying a null mutation in ift122 encoding a component of IFT-A. ift122 mutant larvae show unexpectedly mild phenotypes, compared with those of mutants defective in IFT-B. ift122 mutants exhibit a slow onset of progressive photoreceptor degeneration mainly after 7 days post-fertilization. ift122 mutant larvae also develop cystic kidney but not curly body, both of which are typically observed in various ciliary mutants. ift122 mutants display a loss of cilia in the inner ear hair cells and nasal pit epithelia. Loss of ift122 causes disorganization of outer segment discs. Ectopic accumulation of an IFT-B component, ift88, is observed in the ift122 mutant photoreceptor cilia. In addition, pulse-chase experiments using GFP-opsin fusion proteins revealed that ift122 is required for the efficient transport of opsin and the distal elongation of outer segments. These results show that IFT-A is essential for the efficient transport of outer segment proteins, including opsin, and for the survival of retinal photoreceptor cells, rendering the ift122 mutant a unique model for human retinal degenerative diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Loss of ift122, a Retrograde Intraflagellar Transport (IFT) Complex Component, Leads to Slow, Progressive Photoreceptor Degeneration Due to Inefficient Opsin Transport*

PubMed Central

Boubakri, Meriam; Chaya, Taro; Hirata, Hiromi; Kajimura, Naoko; Kuwahara, Ryusuke; Ueno, Akiko; Malicki, Jarema; Furukawa, Takahisa; Omori, Yoshihiro

2016-01-01

In the retina, aberrant opsin transport from cell bodies to outer segments leads to retinal degenerative diseases such as retinitis pigmentosa. Opsin transport is facilitated by the intraflagellar transport (IFT) system that mediates the bidirectional movement of proteins within cilia. In contrast to functions of the anterograde transport executed by IFT complex B (IFT-B), the precise functions of the retrograde transport mediated by IFT complex A (IFT-A) have not been well studied in photoreceptor cilia. Here, we analyzed developing zebrafish larvae carrying a null mutation in ift122 encoding a component of IFT-A. ift122 mutant larvae show unexpectedly mild phenotypes, compared with those of mutants defective in IFT-B. ift122 mutants exhibit a slow onset of progressive photoreceptor degeneration mainly after 7 days post-fertilization. ift122 mutant larvae also develop cystic kidney but not curly body, both of which are typically observed in various ciliary mutants. ift122 mutants display a loss of cilia in the inner ear hair cells and nasal pit epithelia. Loss of ift122 causes disorganization of outer segment discs. Ectopic accumulation of an IFT-B component, ift88, is observed in the ift122 mutant photoreceptor cilia. In addition, pulse-chase experiments using GFP-opsin fusion proteins revealed that ift122 is required for the efficient transport of opsin and the distal elongation of outer segments. These results show that IFT-A is essential for the efficient transport of outer segment proteins, including opsin, and for the survival of retinal photoreceptor cells, rendering the ift122 mutant a unique model for human retinal degenerative diseases. PMID:27681595

Further Insights into the Ciliary Gene and Protein KIZ and Its Murine Ortholog PLK1S1 Mutated in Rod-Cone Dystrophy

PubMed Central

Méjécase, Cécile; Bertelli, Matteo; Terray, Angélique; Michiels, Christelle; Condroyer, Christel; Fouquet, Stéphane; Sadoun, Maxime; Clérin, Emmanuelle; Liu, Binqian; Léveillard, Thierry; Goureau, Olivier; Sahel, José-Alain; Audo, Isabelle

2017-01-01

We identified herein additional patients with rod-cone dystrophy (RCD) displaying mutations in KIZ, encoding the ciliary centrosomal protein kizuna and performed functional characterization of the respective protein in human fibroblasts and of its mouse ortholog PLK1S1 in the retina. Mutation screening was done by targeted next generation sequencing and subsequent Sanger sequencing validation. KIZ mRNA levels were assessed on blood and serum-deprived human fibroblasts from a control individual and a patient, compound heterozygous for the c.52G>T (p.Glu18*) and c.119_122del (p.Lys40Ilefs*14) mutations in KIZ. KIZ localization, documentation of cilium length and immunoblotting were performed in these two fibroblast cell lines. In addition, PLK1S1 immunolocalization was conducted in mouse retinal cryosections and isolated rod photoreceptors. Analyses of additional RCD patients enabled the identification of two homozygous mutations in KIZ, the known c.226C>T (p.Arg76*) mutation and a novel variant, the c.3G>A (p.Met1?) mutation. Albeit the expression levels of KIZ were three-times lower in the patient than controls in whole blood cells, further analyses in control- and mutant KIZ patient-derived fibroblasts unexpectedly revealed no significant difference between the two genotypes. Furthermore, the averaged monocilia length in the two fibroblast cell lines was similar, consistent with the preserved immunolocalization of KIZ at the basal body of the primary cilia. Analyses in mouse retina and isolated rod photoreceptors showed PLK1S1 localization at the base of the photoreceptor connecting cilium. In conclusion, two additional patients with mutations in KIZ were identified, further supporting that defects in KIZ/PLK1S1, detected at the basal body of the primary cilia in fibroblasts, and the photoreceptor connecting cilium in mouse, respectively, are involved in RCD. However, albeit the mutations were predicted to lead to nonsense mediated mRNA decay, we could not detect changes upon expression levels, protein localization or cilia length in KIZ-mutated fibroblast cells. Together, our findings unveil the limitations of fibroblasts as a cellular model for RCD and call for other models such as induced pluripotent stem cells to shed light on retinal pathogenic mechanisms of KIZ mutations. PMID:29057815

Mutations in ARL2BP, Encoding ADP-Ribosylation-Factor-Like 2 Binding Protein, Cause Autosomal-Recessive Retinitis Pigmentosa

PubMed Central

Davidson, Alice E.; Schwarz, Nele; Zelinger, Lina; Stern-Schneider, Gabriele; Shoemark, Amelia; Spitzbarth, Benjamin; Gross, Menachem; Laxer, Uri; Sosna, Jacob; Sergouniotis, Panagiotis I.; Waseem, Naushin H.; Wilson, Robert; Kahn, Richard A.; Plagnol, Vincent; Wolfrum, Uwe; Banin, Eyal; Hardcastle, Alison J.; Cheetham, Michael E.; Sharon, Dror; Webster, Andrew R.

2013-01-01

Retinitis pigmentosa (RP) is a genetically heterogeneous retinal degeneration characterized by photoreceptor death, which results in visual failure. Here, we used a combination of homozygosity mapping and exome sequencing to identify mutations in ARL2BP, which encodes an effector protein of the small GTPases ARL2 and ARL3, as causative for autosomal-recessive RP (RP66). In a family affected by RP and situs inversus, a homozygous, splice-acceptor mutation, c.101−1G>C, which alters pre-mRNA splicing of ARLBP2 in blood RNA, was identified. In another family, a homozygous c.134T>G (p.Met45Arg) mutation was identified. In the mouse retina, ARL2BP localized to the basal body and cilium-associated centriole of photoreceptors and the periciliary extension of the inner segment. Depletion of ARL2BP caused cilia shortening. Moreover, depletion of ARL2, but not ARL3, caused displacement of ARL2BP from the basal body, suggesting that ARL2 is vital for recruiting or anchoring ARL2BP at the base of the cilium. This hypothesis is supported by the finding that the p.Met45Arg amino acid substitution reduced binding to ARL2 and caused the loss of ARL2BP localization at the basal body in ciliated nasal epithelial cells. These data demonstrate a role for ARL2BP and ARL2 in primary cilia function and that this role is essential for normal photoreceptor maintenance and function. PMID:23849777

Comparative Proteomics Reveals Timely Transport into Cilia of Regulators or Effectors as a Mechanism Underlying Ciliary Disassembly.

PubMed

Wang, Limei; Gu, Lixiao; Meng, Dan; Wu, Qiong; Deng, Haiteng; Pan, Junmin

2017-07-07

Primary cilia are assembled and disassembled during cell cycle progression. During ciliary disassembly, ciliary axonemal microtubules (MTs) are depolymerized accompanied by extensive posttranslational protein modifications of ciliary proteins including protein phosphorylation, methylation, and ubiquitination. These events are hypothesized to involve transport of effectors or regulators into cilia at the time of ciliary disassembly from the cell body. To prove this hypothesis and identify new proteins involved in ciliary disassembly, we analyzed disassembling flagella in Chlamydomonas using comparative proteomics with TMT labeling. Ninety-one proteins were found to increase more than 1.4-fold in four replicates. The proteins of the IFT machinery not only increase but also exhibit stoichiometric changes. The other proteins that increase include signaling molecules, chaperones, and proteins involved in microtubule dynamics or stability. In particular, we have identified a ciliopathy protein C21orf2, the AAA-ATPase CDC48, that is involved in segregating polypeptides from large assemblies or cellular structures, FAP203 and FAP236, which are homologous to stabilizers of axonemal microtubules. Our data demonstrate that ciliary transport of effectors or regulators is one of the mechanisms underlying ciliary disassembly. Further characterization of the proteins identified will provide new insights into our understanding of ciliary disassembly and likely ciliopathy.

Odorants selectively activate distinct G protein subtypes in olfactory cilia.

PubMed

Schandar, M; Laugwitz, K L; Boekhoff, I; Kroner, C; Gudermann, T; Schultz, G; Breer, H

1998-07-03

Chemoelectrical signal transduction in olfactory neurons appears to involve intracellular reaction cascades mediated by heterotrimeric GTP-binding proteins. In this study attempts were made to identify the G protein subtype(s) in olfactory cilia that are activated by the primary (odorant) signal. Antibodies directed against the alpha subunits of distinct G protein subtypes interfered specifically with second messenger reponses elicited by defined subsets of odorants; odor-induced cAMP-formation was attenuated by Galphas antibodies, whereas Galphao antibodies blocked odor-induced inositol 1,4, 5-trisphosphate (IP3) formation. Activation-dependent photolabeling of Galpha subunits with [alpha-32P]GTP azidoanilide followed by immunoprecipitation using subtype-specific antibodies enabled identification of particular individual G protein subtypes that were activated upon stimulation of isolated olfactory cilia by chemically distinct odorants. For example odorants that elicited a cAMP response resulted in labeling of a Galphas-like protein, whereas odorants that elicited an IP3 response led to the labeling of a Galphao-like protein. Since odorant-induced IP3 formation was also blocked by Gbeta antibodies, activation of olfactory phospholipase C might be mediated by betagamma subunits of a Go-like G protein. These results indicate that different subsets of odorants selectively trigger distinct reaction cascades and provide evidence for dual transduction pathways in olfactory signaling.

Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation

PubMed Central

Vladar, Eszter K.; Nayak, Jayakar V.; Milla, Carlos E.; Axelrod, Jeffrey D.

2016-01-01

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. PMID:27570836

Role of spatial heterogeneity in the collective dynamics of cilia beating in a minimal one-dimensional model

NASA Astrophysics Data System (ADS)

Dey, Supravat; Massiera, Gladys; Pitard, Estelle

2018-01-01

Cilia are elastic hairlike protuberances of the cell membrane found in various unicellular organisms and in several tissues of most living organisms. In some tissues such as the airway tissues of the lung, the coordinated beating of cilia induces a fluid flow of crucial importance as it allows the continuous cleaning of our bronchia, known as mucociliary clearance. While most of the models addressing the question of collective dynamics and metachronal wave consider homogeneous carpets of cilia, experimental observations rather show that cilia clusters are heterogeneously distributed over the tissue surface. The purpose of this paper is to investigate the role of spatial heterogeneity on the coherent beating of cilia using a very simple one-dimensional model for cilia known as the rower model. We systematically study systems consisting of a few rowers to hundreds of rowers and we investigate the conditions for the emergence of collective beating. When considering a small number of rowers, a phase drift occurs, hence, a bifurcation in beating frequency is observed as the distance between rower clusters is changed. In the case of many rowers, a distribution of frequencies is observed. We found in particular the pattern of the patchy structure that shows the best robustness in collective beating behavior, as the density of cilia is varied over a wide range.

Mathematical embryology: the fluid mechanics of nodal cilia

NASA Astrophysics Data System (ADS)

Smith, D. J.; Smith, A. A.; Blake, J. R.

2011-07-01

Left-right symmetry breaking is critical to vertebrate embryonic development; in many species this process begins with cilia-driven flow in a structure termed the `node'. Primary `whirling' cilia, tilted towards the posterior, transport morphogen-containing vesicles towards the left, initiating left-right asymmetric development. We review recent theoretical models based on the point-force stokeslet and point-torque rotlet singularities, explaining how rotation and surface-tilt produce directional flow. Analysis of image singularity systems enforcing the no-slip condition shows how tilted rotation produces a far-field `stresslet' directional flow, and how time-dependent point-force and time-independent point-torque models are in this respect equivalent. Associated slender body theory analysis is reviewed; this approach enables efficient and accurate simulation of three-dimensional time-dependent flow, time-dependence being essential in predicting features of the flow such as chaotic advection, which have subsequently been determined experimentally. A new model for the nodal flow utilising the regularized stokeslet method is developed, to model the effect of the overlying Reichert's membrane. Velocity fields and particle paths within the enclosed domain are computed and compared with the flow profiles predicted by previous `membrane-less' models. Computations confirm that the presence of the membrane produces flow-reversal in the upper region, but no continuous region of reverse flow close to the epithelium. The stresslet far-field is no longer evident in the membrane model, due to the depth of the cavity being of similar magnitude to the cilium length. Simulations predict that vesicles released within one cilium length of the epithelium are generally transported to the left via a `loopy drift' motion, sometimes involving highly unpredictable detours around leftward cilia [truncated

Effects of theophylline on expression of the long cilia phenotype in sand dollar blastulae.

PubMed

Riederer-Henderson, M A

1988-04-01

Previously, increases in ciliary length have only been obtained through genetic mutation in Chlamydomonas or by incubation of swimming echinoderm blastulae in trypsin or elastase. We have found that the phenotypic switch from short to long cilia on sand dollar blastulae can also be effected by incubation in theophylline. Cilia detached from control blastulae have a mean length of 21 +/- 7 microns with 10% of the cilia being greater than 30 microns. Upon incubation in 10 mM theophylline additional long cilia appeared after 10 hours and by 24-32 hours 1/2-3/4 of the embryo was covered with long cilia. The percentage of long cilia increased to 65% with a mean length of 40.0 +/- 17.6 microns. Incubation in other methylxanthines, such as aminophylline, caffeine, or isobutylmethylxanthine, inhibited development but had no effect on ciliary length distribution. Dibutyryl cAMP, 8-bromoadenosine, and calcium ionophore also had no effect on ciliary length. Cyclic AMP levels were measured and showed only slight differences among controls and embryos incubated in trypsin, caffeine, or theophylline. These data suggest that theophylline may be altering ciliary length control through some mechanism other than elevations in cAMP.

Pneumatically-actuated artificial cilia array for biomimetic fluid propulsion.

PubMed

Gorissen, Benjamin; de Volder, Michaël; Reynaerts, Dominiek

2015-11-21

Arrays of beating cilia emerged in nature as one of the most efficient propulsion mechanisms at a small scale, and are omnipresent in microorganisms. Previous attempts at mimicking these systems have foundered against the complexity of fabricating small-scale cilia exhibiting complex beating motions. In this paper, we propose for the first time arrays of pneumatically-actuated artificial cilia that are able to address some of these issues. These artificial cilia arrays consist of six highly flexible silicone rubber actuators with a diameter of 1 mm and a length of 8 mm that can be actuated independently from each other. In an experimental setup, the effects of the driving frequency, phase difference and duty cycle on the net flow in a closed-loop channel have been studied. Net fluid speeds of up to 19 mm s(-1) have been measured. Further, it is possible to invert the flow direction by simply changing the driving frequency or by changing the duty cycle of the driving block pulse pressure wave without changing the bending direction of the cilia. Using PIV measurements, we corroborate for the first time existing mathematical models of cilia arrays to measurements on prototypes.

Cilia driven flow networks in the brain

NASA Astrophysics Data System (ADS)

Wang, Yong; Faubel, Regina; Westendorf, Chrsitian; Eichele, Gregor; Bodenschatz, Eberhard

Neurons exchange soluble substances via the cerebrospinal fluid (CSF) that fills the ventricular system. The walls of the ventricular cavities are covered with motile cilia that constantly beat and thereby induce a directional flow. We recently discovered that cilia in the third ventricle generate a complex flow pattern leading to partitioning of the ventricular volume and site-directed transport paths along the walls. Transient and daily recurrent alterations in the cilia beating direction lead to changes in the flow pattern. This has consequences for delivery of CSF components along the near wall flow. The contribution of this cilia-induced flow to overall CSF flow remains to be investigated. The state-of-art lattice Boltzmann method is adapted for studying the CFS flow. The 3D geometry of the third ventricle at high resolution was reconstructed. Simulation of CSF flow without cilia in this geometry confirmed that the previous idea about unidirectional flow does not explain how different components of CSF can be delivered to their various target sites. We study the contribution of the cilia-induced flow pattern to overall CSF flow and identify target areas for site-specific delivery of CSF-constituents with respect to the temporal changes.

[The effect of 18β-sodium glycyrrhetinic acid on the nasal mucosa epithelial cilia in rat models of allergic rhinitis].

PubMed

Yang, Jing; Xi, Kehu; Gui, Yan; Wang, Youhu; Zhang, Fuhong; Ma, Chunxia; Hong, Hao; Liu, Xiangyi; Meng, Nannan; Zhang, Xiaobing

2015-12-01

To investigate 18β-sodium glycyrrhetinic acid impact on nasal mucosa epithelial cilia in rat models of allergic rhinitis (AR). AR models were established by ovalbumin-induction. Wister rats were randomly divided into groups as normal group, model group, budesonide (0.2 mg/kg) group and sodium glycyrrhetinic acid (20 mg/kg and 40 mg/kg) group after the success of AR models. At 2 weeks and 4 weeks after treatment, the behavioral changes of rats were observed and recorded, and nasal septum mucosae were collected after 2 week and 4 week intervention, and the morphological changes of nasal mucosae were observed by electron microscope. Model group developed typical AR symptoms, the total score in all animals was > 5. With budesonide and sodium glycyrrhetinic acid treatment, the AR symptoms were relieved, and the total scores were reduced significantly (P < 0.01). Compared with the model group: after 2 weeks' intervention, thick mucous secretions on the top of columnar epithelium cilia in rat nasal mucosa was significantly reduced, and cilia adhesion, lodging, shedding were relieved in budesonide group and sodium glycyrrhetinic acid group, the relieve in budesonide group was slightly better than that in sodium glycyrrhetinic acid group; after 4 week intervention, Cilia adhesion, lodging, shedding were completely vanished, and the cilia were ranged in regular direction in budesonide group and sodium glycyrrhetinic acid group. Cilia in sodium glycyrrhetinic acid (20 mg/kg) group was more orderly, smooth than that in budesonide group and sodium glycyrrhetinic acid group (40 mg/kg), and the condition of cilia in sodium glycyrrhetinic acid group (20 mg/kg) was similar to the normal group. 18β-sodium glycyrrhetinic acid is effective to restrain the pathological changes of nasal mucosa cilia in rat models of AR.

Hydrodynamic interactions of cilia on a spherical body

NASA Astrophysics Data System (ADS)

Nasouri, Babak; Elfring, Gwynn J.

2015-11-01

The emergence of metachronal waves in ciliated microorganisms can arise solely from the hydrodynamic interactions between the cilia. For a chain of cilia attached to a flat ciliate, it was observed that fluid forces can lead the system to form a metachronal wave. However, several microorganisms such as paramecium and volvox possess a curved shaped ciliate body. To understand the effect of this geometry on the formation of metachronal waves, we evaluate the hydrodynamic interactions of cilia near a large spherical body. Using a minimal model, we show that for a chain of cilia around the sphere, the embedded periodicity in the geometry leads the system to synchronize. We also report an emergent wave-like behavior when an asymmetry is introduced to the system.

Left-right asymmetry: cilia and calcium revisited.

PubMed

Blum, Martin; Vick, Philipp

2015-03-02

Leftward flow generated by motile cilia is known to underlie left-right asymmetry in vertebrate embryos. A new study now links intraciliary calcium oscillations to cilia motility and the downstream nodal signaling cascade that drives left-sided development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fluid pumping using magnetic cilia

NASA Astrophysics Data System (ADS)

Hanasoge, Srinivas; Ballard, Matt; Alexeev, Alexander; Hesketh, Peter; Woodruff School of Mechanical Engineering Team

2016-11-01

Using experiments and computer simulations, we examine fluid pumping by artificial magnetic cilia fabricated using surface micromachining techniques. An asymmetry in forward and recovery strokes of the elastic cilia causes the net pumping in a creeping flow regime. We show this asymmetry in the ciliary strokes is due to the change in magnetization of the elastic cilia combined with viscous force due to the fluid. Specifically, the time scale for forward stroke is mostly governed by the magnetic forces, whereas the time scale for the recovery stroke is determined by the elastic and viscous forces. These different time scales result in different cilia deformation during forward and backward strokes which in turn lead to the asymmetry in the ciliary motion. To disclose the physics of magnetic cilia pumping we use a hybrid lattice Boltzmann and lattice spring method. We validate our model by comparing the simulation results with the experimental data. The results of our study will be useful to design microfluidic systems for fluid mixing and particle manipulation including different biological particles. USDA and NSF.

Dampened Hedgehog signaling but normal Wnt signaling in zebrafish without cilia

PubMed Central

Huang, Peng; Schier, Alexander F.

2009-01-01

Summary Cilia have been implicated in Hedgehog (Hh) and Wnt signaling in mouse but not in Drosophila. To determine whether the role of cilia is conserved in zebrafish, we generated maternal-zygotic (MZ) oval (ovl; ift88) mutants that lack all cilia. MZovl mutants display normal canonical and non-canonical Wnt signaling but show defects in Hh signaling. As in mouse, zebrafish cilia are required to mediate the activities of Hh, Ptc, Smo and PKA. However, in contrast to mouse Ift88 mutants, which show a dramatic reduction in Hh signaling, zebrafish MZovl mutants display dampened, but expanded, Hh pathway activity. This activity is largely due to gli1, the expression of which is fully dependent on Hh signaling in mouse but not in zebrafish. These results reveal a conserved requirement for cilia in transducing the activity of upstream regulators of Hh signaling but distinct phenotypic effects due to differential regulation and differing roles of transcriptional mediators. PMID:19700616

Construction of artificial cilia from microtubules and kinesins through a well-designed bottom-up approach.

PubMed

Sasaki, Ren; Kabir, Arif Md Rashedul; Inoue, Daisuke; Anan, Shizuka; Kimura, Atsushi P; Konagaya, Akihiko; Sada, Kazuki; Kakugo, Akira

2018-04-05

Self-organized structures of biomolecular motor systems, such as cilia and flagella, play key roles in the dynamic processes of living organisms, like locomotion or the transportation of materials. Although fabrication of such self-organized structures from reconstructed biomolecular motor systems has attracted much attention in recent years, a systematic construction methodology is still lacking. In this work, through a bottom-up approach, we fabricated artificial cilia from a reconstructed biomolecular motor system, microtubule/kinesin. The artificial cilia exhibited a beating motion upon the consumption, by the kinesins, of the chemical energy obtained from the hydrolysis of adenosine triphosphate (ATP). Several design parameters, such as the length of the microtubules, the density of the kinesins along the microtubules, the depletion force among the microtubules, etc., have been identified, which permit tuning of the beating frequency of the artificial cilia. The beating frequency of the artificial cilia increases upon increasing the length of the microtubules, but declines for the much longer microtubules. A high density of the kinesins along the microtubules is favorable for the beating motion of the cilia. The depletion force induced bundling of the microtubules accelerated the beating motion of the artificial cilia and increased the beating frequency. This work helps understand the role of self-assembled structures of the biomolecular motor systems in the dynamics of living organisms and is expected to expedite the development of artificial nanomachines, in which the biomolecular motors may serve as actuators.

Basal body stability and ciliogenesis requires the conserved component Poc1

PubMed Central

Osborn, Daniel P.S.; Giddings, Thomas H.; Beales, Philip L.; Winey, Mark

2009-01-01

Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain–containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T. thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis. PMID:20008567

VDAC3 and Mps1 negatively regulate ciliogenesis.

PubMed

Majumder, Shubhra; Fisk, Harold A

2013-03-01

Centrosomes serve to organize new centrioles in cycling cells, whereas in quiescent cells they assemble primary cilia. We have recently shown that the mitochondrial porin VDAC3 is also a centrosomal protein that is predominantly associated with the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes. Here, we show that depletion of VDAC3 causes inappropriate ciliogenesis in cycling cells, while expression of GFP-VDAC3 suppresses ciliogenesis in quiescent cells. Mps1 also negatively regulates ciliogenesis, and the inappropriate ciliogenesis caused by VDAC3 depletion can be bypassed by targeting Mps1 to centrosomes independently of VDAC3. Thus, our data show that a VDAC3-Mps1 module at the centrosome promotes ciliary disassembly during cell cycle entry and suppresses cilia assembly in proliferating cells. Our data also suggests that VDAC3 might be a link between mitochondrial dysfunction and ciliopathies in mammalian cells.

Stimulation of Electro-Olfactogram Responses in the Main Olfactory Epithelia by Airflow Depend on the Type 3 Adenylyl Cyclase

PubMed Central

Chen, Xuanmao; Xia, Zhengui; Storm, Daniel R.

2012-01-01

Cilia of olfactory sensory neurons (OSN) are the primary sensory organelles for olfaction. The detection of odorants by the main olfactory epithelium (MOE) depends on coupling of odorant receptors to the type 3 adenylyl cyclase (AC3) in olfactory cilia. We monitored the effect of airflow on electro-olfactogram (EOG) responses and found that the MOE of mice can sense mechanical forces generated by airflow. The airflow-sensitive EOG response in the MOE was attenuated when cAMP was increased by odorants or by forskolin suggesting a common mechanism for airflow and odorant detection. In addition, the sensitivity to airflow was significantly impaired in the MOE from AC3−/− mice. We conclude that AC3 in the MOE is required for detecting the mechanical force of airflow, which in turn may regulate odorant perception during sniffing. PMID:23136416

New ADCY3 Variants Dance in Obesity Etiology.

PubMed

Tian, Yan; Peng, Boqiang; Fu, Xianghui

2018-02-14

The genetic etiology for obesity-related traits remains elusive. Recent studies link novel ADCY3 variants to obesity and diabetes, and identify an important role of ADCY3-mediated signaling at neuronal primary cilia in the predisposition of obesity. These findings provide new information on obesity etiology and suggest potential anti-obesity therapeutic strategies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Primary Events in Olfactory Receptiom

DTIC Science & Technology

1989-10-10

phase after extraction with Triton X-1 14 and does not bind either concanavalin A or wheat germ agglutinin. When phosphorylation is allowed to proceed in...a low basal level of endogenous activity. Phosphorylation is reversible by treatment of phosphorylated cilia with alkaline phosphatase . Comparing...not with the non-phosphorylated peptide or individual phosphorylated amino acids , such as phosphoserine and phosphotyrosine. We will use these antisera

Synchronization and Collective Dynamics of Flagella and Cilia as Hydrodynamically Coupled Oscillators

NASA Astrophysics Data System (ADS)

Uchida, Nariya; Golestanian, Ramin; Bennett, Rachel R.

2017-10-01

Cooperative motion of flagella and cilia faciliates swimming of microorganisms and material transport in the body of multicellular organisms. Using minimal models, we address the roles of hydrodynamic interaction in synchronization and collective dynamics of flagella and cilia. Collective synchronization of bacterial flagella is studied with a model of bacterial carpets. Cilia and eukaryotic flagella are characterized by periodic modulation of their driving forces, which produces various patterns of two-body synchronization and metachronal waves. Long-range nature of the interaction introduces novel features in the dynamics of these model systems. The flagella of a swimmer synchronize also by a viscous drag force mediated through the swimmer's body. Recent advance in experimental studies of the collective dynamics of flagella, cilia and related artificial systems are summarized.

Metachronal wave of artificial cilia array actuated by applied magnetic field

NASA Astrophysics Data System (ADS)

Tsumori, Fujio; Marume, Ryuma; Saijou, Akinori; Kudo, Kentaro; Osada, Toshiko; Miura, Hideshi

2016-06-01

In this paper, a biomimetic microstructure related to cilia, which are effective fluidic and conveying systems in nature, is described. Authors have already reported that a magnetic elastomer pillar actuated by a rotating magnetic field can work like a natural cilium. In the present work, we show examples of a cilia array with a metachronal wave as the next step. A metachronal wave is a sequential action of a number of cilia. It is theoretically known that a metachronal wave gives a higher fluidic efficiency; however, there has been no report on a metachronal wave by artificial cilia. We prepared magnetic elastomer pillars that contain chainlike clusters of magnetic particles. The orientation of chains was set to be different in each pillar so that each pillar will deform with a different phase.

Variations in the ultrastructure of human nasal cilia including abnormalities found in retinitis pigmentosa.

PubMed Central

Fox, B; Bull, T B; Arden, G B

1980-01-01

The electron microscopic structure of cilia from the inferior turbinate of the nose was studied in 12 adults, four with chronic sinusitis, one with allergic rhinitis, two with bronchiectasis, three with deviated nasal septum, and two normals. The changes are compared with those found in nasal cilia in 14 patients with retinitis pigmentosa. There were compound cilia in the seven cases with chronic sinusitis, allergic rhinitis, and bronchiectasis but, apart from this, the structure of the cilia was similar in all 12 cases. There were variations in the microtubular pattern in about 4% of cilia, dynein arms were not seen in 4%, and in the rest an average of 5-6 dynein arms were seen in each cilium. The orientation of the cilia was 0 to 90 degrees. In the retinitis pigmentosa patients there was a highly significant increase in cilial abnormalities. The establishment on a quantitative basis of the variations in normal structure of nasal cilila facilitated the recognition of an association between cilial abnormalities and retinitis pigmentosa and should help in the identification of associations that may exist between cilial abnormalities and other diseases. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 7 Fig. 8 PMID:7400333

[Bardet-Biedl syndrome: cilia and obesity - from genes to integrative approaches].

PubMed

Chennen, Kirsley; Scerbo, Maria Julia; Dollfus, Hélène; Poch, Olivier; Marion, Vincent

2014-11-01

The primary cilium is a specialized organelle, present at the surface of most eukaryotic cells, whose main function is to detect, integrate and transmit intra- and extra-cellular signals. Its dysfunction usually results in a group of severe clinical manifestations nowadays termed ciliopathies. The latter can be of syndromic nature with multi-organ dysfunctions and can also be associated with a morbid obese phenotype, like it is the case in the iconic ciliopathy, the Bardet Biedl syndrome (BBS). This review will discuss the contribution of the unique context offered by the emblematic BBS for understanding the mechanisms leading to obesity via the involvement of the primary cilium together with identification of novel molecular players and signaling pathways it has helped to highlight. In the current context of translational medicine and system biology, this article will also discuss the potential benefits and challenges posed by these techniques via multi-level approaches to better dissect the underlying mechanisms leading to the complex condition of obesity. © 2014 médecine/sciences – Inserm.

Caenorhabditis elegans glutamylating enzymes function redundantly in male mating.

PubMed

Chawla, Daniel G; Shah, Ruchi V; Barth, Zachary K; Lee, Jessica D; Badecker, Katherine E; Naik, Anar; Brewster, Megan M; Salmon, Timothy P; Peel, Nina

2016-09-15

Microtubule glutamylation is an important modulator of microtubule function and has been implicated in the regulation of centriole stability, neuronal outgrowth and cilia motility. Glutamylation of the microtubules is catalyzed by a family of tubulin tyrosine ligase-like (TTLL) enzymes. Analysis of individual TTLL enzymes has led to an understanding of their specific functions, but how activities of the TTLL enzymes are coordinated to spatially and temporally regulate glutamylation remains relatively unexplored. We have undertaken an analysis of the glutamylating TTLL enzymes in C. elegans We find that although all five TTLL enzymes are expressed in the embryo and adult worm, loss of individual enzymes does not perturb microtubule function in embryonic cell divisions. Moreover, normal dye-filling, osmotic avoidance and male mating behavior indicate the presence of functional amphid cilia and male-specific neurons. A ttll-4(tm3310); ttll-11(tm4059); ttll-5(tm3360) triple mutant, however, shows reduced male mating efficiency due to a defect in the response step, suggesting that these three enzymes function redundantly, and that glutamylation is required for proper function of the male-specific neurons. © 2016. Published by The Company of Biologists Ltd.

Positioning of centrioles is a conserved readout of Frizzled planar cell polarity signalling

PubMed Central

Carvajal-Gonzalez, Jose Maria; Roman, Angel-Carlos; Mlodzik, Marek

2016-01-01

Planar cell polarity (PCP) signalling is a well-conserved developmental pathway regulating cellular orientation during development. An evolutionarily conserved pathway readout is not established and, moreover, it is thought that PCP mediated cellular responses are tissue-specific. A key PCP function in vertebrates is to regulate coordinated centriole/cilia positioning, a function that has not been associated with PCP in Drosophila. Here we report instructive input of Frizzled-PCP (Fz/PCP) signalling into polarized centriole positioning in Drosophila wings. We show that centrioles are polarized in pupal wing cells as a readout of PCP signalling, with both gain and loss-of-function Fz/PCP signalling affecting centriole polarization. Importantly, loss or gain of centrioles does not affect Fz/PCP establishment, implicating centriolar positioning as a conserved PCP-readout, likely downstream of PCP-regulated actin polymerization. Together with vertebrate data, these results suggest a unifying model of centriole/cilia positioning as a common downstream effect of PCP signalling from flies to mammals. PMID:27021213

Positioning of centrioles is a conserved readout of Frizzled planar cell polarity signalling.

PubMed

Carvajal-Gonzalez, Jose Maria; Roman, Angel-Carlos; Mlodzik, Marek

2016-03-29

Planar cell polarity (PCP) signalling is a well-conserved developmental pathway regulating cellular orientation during development. An evolutionarily conserved pathway readout is not established and, moreover, it is thought that PCP mediated cellular responses are tissue-specific. A key PCP function in vertebrates is to regulate coordinated centriole/cilia positioning, a function that has not been associated with PCP in Drosophila. Here we report instructive input of Frizzled-PCP (Fz/PCP) signalling into polarized centriole positioning in Drosophila wings. We show that centrioles are polarized in pupal wing cells as a readout of PCP signalling, with both gain and loss-of-function Fz/PCP signalling affecting centriole polarization. Importantly, loss or gain of centrioles does not affect Fz/PCP establishment, implicating centriolar positioning as a conserved PCP-readout, likely downstream of PCP-regulated actin polymerization. Together with vertebrate data, these results suggest a unifying model of centriole/cilia positioning as a common downstream effect of PCP signalling from flies to mammals.

Mouse models of ciliopathies: the state of the art

PubMed Central

Norris, Dominic P.; Grimes, Daniel T.

2012-01-01

The ciliopathies are an apparently disparate group of human diseases that all result from defects in the formation and/or function of cilia. They include disorders such as Meckel-Grüber syndrome (MKS), Joubert syndrome (JBTS), Bardet-Biedl syndrome (BBS) and Alström syndrome (ALS). Reflecting the manifold requirements for cilia in signalling, sensation and motility, different ciliopathies exhibit common elements. The mouse has been used widely as a model organism for the study of ciliopathies. Although many mutant alleles have proved lethal, continued investigations have led to the development of better models. Here, we review current mouse models of a core set of ciliopathies, their utility and future prospects. PMID:22566558

The mouse homeobox gene Noto regulates node morphogenesis, notochordal ciliogenesis, and left–right patterning

PubMed Central

Beckers, Anja; Alten, Leonie; Viebahn, Christoph; Andre, Philipp; Gossler, Achim

2007-01-01

The mouse homeobox gene Noto represents the homologue of zebrafish floating head (flh) and is expressed in the organizer node and in the nascent notochord. Previous analyses suggested that Noto is required exclusively for the formation of the caudal part of the notochord. Here, we show that Noto is also essential for node morphogenesis, controlling ciliogenesis in the posterior notochord, and the establishment of laterality, whereas organizer functions in anterior–posterior patterning are apparently not compromised. In mutant embryos, left–right asymmetry of internal organs and expression of laterality markers was randomized. Mutant posterior notochord regions were variable in size and shape, cilia were shortened with highly irregular axonemal microtubuli, and basal bodies were, in part, located abnormally deep in the cytoplasm. The transcription factor Foxj1, which regulates the dynein gene Dnahc11 and is required for the correct anchoring of basal bodies in lung epithelial cells, was down-regulated in mutant nodes. Likewise, the transcription factor Rfx3, which regulates cilia growth, was not expressed in Noto mutants, and various other genes important for cilia function or assembly such as Dnahc5 and Nphp3 were down-regulated. Our results establish Noto as an essential regulator of node morphogenesis and ciliogenesis in the posterior notochord, and suggest Noto acts upstream of Foxj1 and Rfx3. PMID:17884984

ATR localizes to the photoreceptor connecting cilium and deficiency leads to severe photoreceptor degeneration in mice.

PubMed

Valdés-Sánchez, Lourdes; De la Cerda, Berta; Diaz-Corrales, Francisco J; Massalini, Simone; Chakarova, Christina F; Wright, Alan F; Bhattacharya, Shomi S

2013-04-15

Ataxia-telangiectasia and Rad3 (ATR), a sensor of DNA damage, is associated with the regulation and control of cell division. ATR deficit is known to cause Seckel syndrome, characterized by severe proportionate short stature and microcephaly. We used a mouse model for Seckel disease to study the effect of ATR deficit on retinal development and function and we have found a new role for ATR, which is critical for the postnatal development of the photoreceptor (PR) layer in mouse retina. The structural and functional characterization of the ATR(+/s) mouse retinas displayed a specific, severe and early degeneration of rod and cone cells resembling some characteristics of human retinal degenerations. A new localization of ATR in the cilia of PRs and the fact that mutant mice have shorter cilia suggests that the PR degeneration here described results from a ciliary defect.

Transport and Mixing Induced by Beating Cilia in Human Airways

PubMed Central

Chateau, Sylvain; D'Ortona, Umberto; Poncet, Sébastien; Favier, Julien

2018-01-01

The fluid transport and mixing induced by beating cilia, present in the bronchial airways, are studied using a coupled lattice Boltzmann—Immersed Boundary solver. This solver allows the simulation of both single and multi-component fluid flows around moving solid boundaries. The cilia are modeled by a set of Lagrangian points, and Immersed Boundary forces are computed onto these points in order to ensure the no-slip velocity conditions between the cilia and the fluids. The cilia are immersed in a two-layer environment: the periciliary layer (PCL) and the mucus above it. The motion of the cilia is prescribed, as well as the phase lag between two cilia in order to obtain a typical collective motion of cilia, known as metachronal waves. The results obtained from a parametric study show that antiplectic metachronal waves are the most efficient regarding the fluid transport. A specific value of phase lag, which generates the larger mucus transport, is identified. The mixing is studied using several populations of tracers initially seeded into the pericilary liquid, in the mucus just above the PCL-mucus interface, and in the mucus far away from the interface. We observe that each zone exhibits different chaotic mixing properties. The larger mixing is obtained in the PCL layer where only a few beating cycles of the cilia are required to obtain a full mixing, while above the interface, the mixing is weaker and takes more time. Almost no mixing is observed within the mucus, and almost all the tracers do not penetrate the PCL layer. Lyapunov exponents are also computed for specific locations to assess how the mixing is performed locally. Two time scales are introduced to allow a comparison between mixing induced by fluid advection and by molecular diffusion. These results are relevant in the context of respiratory flows to investigate the transport of drugs for patients suffering from chronic respiratory diseases. PMID:29559920

Transport and Mixing Induced by Beating Cilia in Human Airways.

PubMed

Chateau, Sylvain; D'Ortona, Umberto; Poncet, Sébastien; Favier, Julien

2018-01-01

The fluid transport and mixing induced by beating cilia, present in the bronchial airways, are studied using a coupled lattice Boltzmann-Immersed Boundary solver. This solver allows the simulation of both single and multi-component fluid flows around moving solid boundaries. The cilia are modeled by a set of Lagrangian points, and Immersed Boundary forces are computed onto these points in order to ensure the no-slip velocity conditions between the cilia and the fluids. The cilia are immersed in a two-layer environment: the periciliary layer (PCL) and the mucus above it. The motion of the cilia is prescribed, as well as the phase lag between two cilia in order to obtain a typical collective motion of cilia, known as metachronal waves. The results obtained from a parametric study show that antiplectic metachronal waves are the most efficient regarding the fluid transport. A specific value of phase lag, which generates the larger mucus transport, is identified. The mixing is studied using several populations of tracers initially seeded into the pericilary liquid, in the mucus just above the PCL-mucus interface, and in the mucus far away from the interface. We observe that each zone exhibits different chaotic mixing properties. The larger mixing is obtained in the PCL layer where only a few beating cycles of the cilia are required to obtain a full mixing, while above the interface, the mixing is weaker and takes more time. Almost no mixing is observed within the mucus, and almost all the tracers do not penetrate the PCL layer. Lyapunov exponents are also computed for specific locations to assess how the mixing is performed locally. Two time scales are introduced to allow a comparison between mixing induced by fluid advection and by molecular diffusion. These results are relevant in the context of respiratory flows to investigate the transport of drugs for patients suffering from chronic respiratory diseases.

Life without centrioles: cilia in the spotlight.

PubMed

Badano, Jose L; Katsanis, Nicholas

2006-06-30

Centrioles are critical cellular components that form the architectural core of both centrosomes and basal bodies, the nucleating structures of cilia. New work, including a study in this issue (), highlights the unexpected finding that lack of centrioles does not impede development in the fruit fly. Rather, flies reach maturity but then die because their sensory neurons lack cilia.

Bardet-Biedl syndrome: A rare cause of end stage renal disease.

PubMed

Hemachandar, R

2015-01-01

Bardet-Biedl syndrome is a rare autosomal recessive disorder, recently categorized as ciliopathy characterized by dysfunction of primary cilia which results in myriad manifestations in various organ systems. Though renal abnormalities can occur in this syndrome, renal failure is a rare presentation. The author reports a case of 18-year-old female who presented with polydactyly, obesity, retinitis pigmentosa, learning disability and renal failure.

Fabrication and characterization of a magnetic micro-actuator based on deformable Fe-doped PDMS artificial cilium using 3D printing

NASA Astrophysics Data System (ADS)

Liu, Fengli; Alici, Gursel; Zhang, Binbin; Beirne, Stephen; Li, Weihua

2015-03-01

This paper proposes the use of a 3D extrusion printer to fabricate artificial magnetic cilium. The cilia are fabricated using polydimethylsiloxane (PDMS) doped with iron particles so that they remain slender and flexible. They can be driven by a magnetic field to closely mimic the behaviour of biological cilia. Doping iron particles to the polymers has already been done; however, to the best of our knowledge, printing such active and soft magnetic structures has not. The existing methods for manufacturing magnetic polymeric structures are complex and difficult to use for the fabrication of micro-sized high-aspect-ratio cilia. The 3D printing technique we propose here is simple and inexpensive compared to previously suggested fabrication methods. In this study, free-standing magnetic PDMS cilia were fabricated in different sizes up to 5 mm in length and 1 mm in width. The stress-strain curves of the PDMS cilia were experimentally obtained to quantify the effect of the concentration of the iron particles on the modulus of elasticity of the cilia. The higher the iron concentration, the higher the modulus of elasticity. We have quantified the characteristics of the cilia made of 40% w/w iron particles in PDMS. A single cilium (5 × 1 × 0.0035 mm) can output up to 27 μN blocking force under a magnetic field of 160 mT. These cilia can be used as a mixer in lap-on-chip applications and as the anchoring and propulsion legs of endoscopic capsule robots operating within the gastrointestinal tract of humans. Analytical expressions estimating the blocking force are established and compared with the experimental results.

Small heat shock proteins are necessary for heart migration and laterality determination in zebrafish

PubMed Central

Lahvic, Jamie L.; Ji, Yongchang; Marin, Paloma; Zuflacht, Jonah P.; Springel, Mark W.; Wosen, Jonathan E.; Davis, Leigh; Hutson, Lara D.; Amack, Jeffrey D.; Marvin, Martha J.

2013-01-01

Small heat shock proteins (sHsps) regulate cellular functions not only under stress, but also during normal development, when they are expressed in organ-specific patterns. Here we demonstrate that two small heat shock proteins expressed in embryonic zebrafish heart, hspb7 and hspb12, have roles in the development of left-right asymmetry. In zebrafish, laterality is determined by the motility of cilia in Kupffer’s vesicle (KV), where hspb7 is expressed; knockdown of hspb7 causes laterality defects by disrupting the motility of these cilia. In embryos with reduced hspb7, the axonemes of KV cilia have a 9+0 structure, while control embyros have a predominately 9+2 structure. Reduction of either hspb7 or hspb12 alters the expression pattern of genes that propagate the signals that establish left-right asymmetry: the nodal-related gene southpaw (spaw) in the lateral plate mesoderm, and its downstream targets pitx2, lefty1 and lefty2. Partial depletion of hspb7 causes concordant heart, brain and visceral laterality defects, indicating that loss of KV cilia motility leads causes coordinated but randomized laterality. Reducing hspb12 leads to similar alterations in the expression of downstream laterality genes, but at a lower penetrance. Simultaneous reduction of hspb7 and hspb12 randomizes heart, brain and visceral laterality, suggesting that these two genes have partially redundant functions in the establishment of left-right asymmetry. In addition, both hspb7 and hspb12 are expressed in the precardiac mesoderm and in the yolk syncytial layer, which supports the migration and fusion of mesodermal cardiac precursors. In embryos in which the reduction of hspb7 or hspb12 was limited to the yolk, migration defects predominated, suggesting that the yolk expression of these genes rather than heart expression is responsible for the migration defects. PMID:24140541

Nature-inspired polymer actuators for micro-fluidic mixing.

NASA Astrophysics Data System (ADS)

den Toonder, Jaap M. J.; Bos, Femke; de Goede, Judith; Anderson, Patrick

2007-11-01

One particular micro-fluidics manipulation mechanism ``designed'' by nature is that due to a covering of beating cilia over the external surface of micro-organisms (e.g. Paramecium). A cilium can be viewed as a small hair or flexible rod (in protozoa: typical length 10 microns and diameter 0.1 microns) which is attached to the surface. We have developed polymer micro-actuators, made with standard micro-technology processing, which respond to an applied electrical or magnetic field by changing their shape. The shape and size of the polymer actuators mimics that of cilia occurring in nature. Flow visualization experiments show that the cilia can generate substantial fluid velocities, in the order of 1 mm/s. In addition, using specially designed geometrical configurations of the cilia, very efficient mixing is obtained. Since the artificial cilia can be actively controlled using electrical signals, they have exciting applications in micro-fluidic devices.

Fabrication and Manipulation of Ciliary Microrobots with Non-reciprocal Magnetic Actuation

PubMed Central

Kim, Sangwon; Lee, Seungmin; Lee, Jeonghun; Nelson, Bradley J.; Zhang, Li; Choi, Hongsoo

2016-01-01

Magnetically actuated ciliary microrobots were designed, fabricated, and manipulated to mimic cilia-based microorganisms such as paramecia. Full three-dimensional (3D) microrobot structures were fabricated using 3D laser lithography to form a polymer base structure. A nickel/titanium bilayer was sputtered onto the cilia part of the microrobot to ensure magnetic actuation and biocompatibility. The microrobots were manipulated by an electromagnetic coil system, which generated a stepping magnetic field to actuate the cilia with non-reciprocal motion. The cilia beating motion produced a net propulsive force, resulting in movement of the microrobot. The magnetic forces on individual cilia were calculated with various input parameters including magnetic field strength, cilium length, applied field angle, actual cilium angle, etc., and the translational velocity was measured experimentally. The position and orientation of the ciliary microrobots were precisely controlled, and targeted particle transportation was demonstrated experimentally. PMID:27470077

Studies on ciliated epithelia of the human genital tract. I. Swelling of the cilia of Fallopian tube epithelium in organ cultures infected with Mycoplasma hominis.

PubMed Central

Mårdh, P A; Weström, L; von Mecklenburg, C; Hammar, E

1976-01-01

Organ cultures of human Fallopian tubes were infected with Mycoplasma hominis. Scanning and transmission electron microscopy revealed swelling of the cilia of the tubal epithelial cells in infected cultures. In some, the entire cilia were swollen; in others, only the tips. Uninfected cultures kept for up to 7 days showed no structural changes in the cilia or other surface structures. M. hominis multiplied in organ cultures, but not in culture medium without tissue. A practical organ culture technique for the preparation of specimens for electron microscopy is described. Images PMID:1260408

Artificial selection for a secondary sexual character in males of Drosophila silvestris from Hawaii

PubMed Central

Carson, Hampton L.; Teramoto, Linden T.

1984-01-01

Drosophila silvestris is endemic to the rain forests of Hawaii Island. In populations from the northeast side, the male foreleg tibia bears about 100 long cilia; these are used as a brush to stimulate the female during courtship. Cyclical family selection for high and low cilia number was carried out on progeny of lines started from single wild isofemales collected at a single site. Significant response in both directions was obtained; in the third generation the lines differed by a mean of 25 cilia. Despite much effort to save them, both lines died out without further advance; the low cilia number line died after five and the high cilia number line died after nine generations. The data are compatible with the view that the cilia character is polygenic and is maintained in nature by a stabilizing sexual selection that also favors a polygenic heterozygous state. Although selection in these experiments resulted in dysgenesis, such a result is not inevitable. Disorganization and reorganization of such a genetic system following a population bottleneck might be important as a predisposing condition for the emergence of a specifically novel and possibly isolating mode of courtship. PMID:16593479

Directed Fluid Transport with Biomimetic ``Silia'' Arrays

NASA Astrophysics Data System (ADS)

Shields, A. R.; Evans, B. A.; Carstens, B. L.; Falvo, M. R.; Washburn, S.; Superfine, R.

2008-10-01

We present results on the long-range, directed fluid transport produced by the collective beating of arrays of biomimetic ``silia.'' Silia are arrays of free-standing nanorods roughly the size of biological cilia, which we fabricate from a polymer-magnetic nanoparticle composite material. With external permanent magnets we actuate our silia such that their motion mimics the beating of biological cilia. Biological cilia have evolved to produce microscale fluid transport and are increasingly being recognized as critical components in a wide range of biological systems. However, despite much effort cilia generated fluid flows remain an area of active study. In the last decade, cilia-driven fluid flow in the embryonic node of vertebrates has been implicated as the initial left-right symmetry breaking event in these embryos. With silia we generate directional fluid transport by mimicking the tilted conical beating of these nodal cilia and seek to answer open questions about the nature of particle advection in such a system. By seeding fluorescent microparticles into the fluid we have noted the existence of two distinct flow regimes. The fluid flow is directional and coherent above the tips of the silia, while between the silia tips and floor particle motion is complicated and suggestive of chaotic advection.

From single cilia to collective waves in human airway ciliated tissues

NASA Astrophysics Data System (ADS)

Cicuta, Pietro; Chioccioli, Maurizio; Feriani, Luigi; Pellicciotta, Nicola; Kotar, Jurij

I will present experimental results on activity of motile cilia on various scales: from waveforms on individual cilia to the synchronised motion in cilia carpets of airway cells. Model synthetic experiments have given us an understanding of how cilia could couple with each other through forces transmitted by the fluid, and thus coordinate to beat into well organized waves (previous work is reviewed in Annu. Rev. Condens. Matter Phys. 7, 1-26 (2016)). Working with live imaging of airway human cells at the different scales, we can now test whether the biological system satisfies the ``simple'' behavior expected of the fluid flow coupling, or if other factors of mechanical forces transmission need to be accounted for. In general being able to link from the scale of molecular biological activity up to the phenomenology of collective dynamics requires to understand the relevant physical mechanism. This understanding then allows informed diagnostics (and perhaps therapeutic) approaches to a variety of diseases where mucociliary clearance in the airways is compromised. We have started exploring particularly cystic fibrosis, where the rheological properties of the mucus are affected and prevent efficient cilia synchronization. ERC Grant HydroSync.

The Effects of Electronic Cigarette (ECIG)-Generated Aerosol and Conventional Cigarette Smoke on the Mucociliary Transport Velocity (MTV) Using the Bullfrog (R. catesbiana) Palate Paradigm

PubMed Central

Palazzolo, Dominic L.; Nelson, John M.; Ely, Emily A.; Crow, Andrew P.; Distin, James; Kunigelis, Stan C.

2017-01-01

Background: While ECIGs are under scrutiny concerning safety, particularly in reference to the physiological impact that aerosolized ECIG liquid (E-liquid) may have on respiratory tissues, others believe that ECIGs are a “Harm Reduction” alternative to conventional cigarettes. Previous studies investigating ciliated respiratory epithelium indicate that smoking shortens cilia length, reduces cilia beat frequency and disrupts respiratory epithelium, which most likely contributes to the inhibition of mucocilliary clearance. Monitoring mucous clearance of respiratory tissues exposed to ECIG-generated aerosol or conventional cigarette smoke, as indexed by mucous transport velocity (MTV), is one way to gauge the impact aerosol and smoke have on the respiratory tract. Therefore, we designed an experiment to test the effect of ECIG-generated aerosol and smoke on MTV using the frog palate paradigm. Methods: Peristaltic pumps transport ECIG-generated aerosol and conventional cigarette smoke into custom-made chambers containing excised bullfrog palates. MTVs were determined before exposure, immediately after exposure and approximately 1 day following exposure. MTVs were also determined (at the same time points) for palates exposed to air (control). Surface and cross sectional SEM images of palates from all three groups were obtained to support MTV data. Results: The results indicate that ECIG-generated aerosol has a modest inhibitory effect (p < 0.05) on MTV 1 day post-exposure (0.09 ± 0.01) compared to control MTV (0.16 ± 0.03 mm/s). In contrast, smoke completely inhibits MTV from 0.14 ± 0.03 mm/s immediately before exposure to 0.00 mm/sec immediately after exposure and the MTV is unable to recover 1 day later. SEM images of control palates and palates exposed to ECIG-generated aerosol both show cilia throughout their epithelial surface, while some areas of palates exposed to smoke are completely devoid of cilia. Additionally, the epithelial thickness of aerosol-exposed palates appears thicker than control palates while smoke-exposed palates appear to be thinner due to epithelial disruption. Conclusions: These results indicate that ECIG-generated aerosol has only a modest effect on mucocilary clearance of bullfrog palates and aerosol sedimentation accounts for epithelial thickening. In accordance with the primary literature, conventional cigarette smoke dramatically inhibits mucociliary clearance and is, in part, due to decreased number of cilia and disruption of the smoke-exposed epithelium. PMID:29321743

Artificial Muscle (AM) Cilia Array for Underwater Systems

DTIC Science & Technology

2016-12-15

structures, including cilia-like structures. Specifically, a custom 3D printer was created that utilizes custom-made Nafion filament for 30 printing of custom... printing ) of IPMC material to create custom-shaped AM structures, including cilia-like structures. Various custom-shaped AM structures were fabricated via...integrating square cross-section IPMC actuators with a printed circuit board power delivery system. IV. Concise Accomplishments Performance

A novel zf-MYND protein, CHB-3, mediates guanylyl cyclase localization to sensory cilia and controls body size of Caenorhabditis elegans.

PubMed

Fujiwara, Manabi; Teramoto, Takayuki; Ishihara, Takeshi; Ohshima, Yasumi; McIntire, Steven L

2010-11-24

Cilia are important sensory organelles, which are thought to be essential regulators of numerous signaling pathways. In Caenorhabditis elegans, defects in sensory cilium formation result in a small-body phenotype, suggesting the role of sensory cilia in body size determination. Previous analyses suggest that lack of normal cilia causes the small-body phenotype through the activation of a signaling pathway which consists of the EGL-4 cGMP-dependent protein kinase and the GCY-12 receptor-type guanylyl cyclase. By genetic suppressor screening of the small-body phenotype of a cilium defective mutant, we identified a chb-3 gene. Genetic analyses placed chb-3 in the same pathway as egl-4 and gcy-12 and upstream of egl-4. chb-3 encodes a novel protein, with a zf-MYND motif and ankyrin repeats, that is highly conserved from worm to human. In chb-3 mutants, GCY-12 guanylyl cyclase visualized by tagged GFP (GCY-12::GFP) fails to localize to sensory cilia properly and accumulates in cell bodies. Our analyses suggest that decreased GCY-12 levels in the cilia of chb-3 mutants may cause the suppression of the small-body phenotype of a cilium defective mutant. By observing the transport of GCY-12::GFP particles along the dendrites to the cilia in sensory neurons, we found that the velocities and the frequencies of the particle movement are decreased in chb-3 mutant animals. How membrane proteins are trafficked to cilia has been the focus of extensive studies in vertebrates and invertebrates, although only a few of the relevant proteins have been identified. Our study defines a new regulator, CHB-3, in the trafficking process and also shows the importance of ciliary targeting of the signaling molecule, GCY-12, in sensory-dependent body size regulation in C. elegans. Given that CHB-3 is highly conserved in mammal, a similar system may be used in the trafficking of signaling proteins to the cilia of other species.

Centrioles are freed from cilia by severing prior to mitosis.

PubMed

Parker, Jeremy D K; Hilton, Laura K; Diener, Dennis R; Rasi, M Qasim; Mahjoub, Moe R; Rosenbaum, Joel L; Quarmby, Lynne M

2010-07-01

Cilia are necessary for normal tissue development and homeostasis and are generally present during interphase, but not in mitosis. The precise mechanism of premitotic ciliary loss has been controversial, with data supporting either sequential disassembly through the transition zone or, alternatively, a severing event at the base of the cilia. Here we show by live cell imaging and immunofluorescence microscopy that resorbing flagella of Chlamydomonas leave remnants associated with the mother cell wall. We postulated that the remnants are the product of severing of doublet microtubules between the basal bodies and the flagellar transition zone, thereby freeing the centrioles to participate in spindle organization. We show via TEM that flagellar remnants are indeed flagellar transition zones encased in vesicles derived from the flagellar membrane. This transition zone vesicle can be lodged within the cell wall or it can be expelled into the environment. This process is observable in Chlamydomonas, first because the released flagellar remnants can remain associated with the cell by virtue of attachments to the cell wall, and second because the Chlamydomonas transition zone is particularly rich with electron-dense structure. However, release of basal bodies for spindle-associated function is likely to be conserved among the eukaryotes. 2010 Wiley-Liss, Inc.

Primary Ciliary Dyskinesia Caused by Homozygous Mutation in DNAL1, Encoding Dynein Light Chain 1

PubMed Central

Mazor, Masha; Alkrinawi, Soliman; Chalifa-Caspi, Vered; Manor, Esther; Sheffield, Val C.; Aviram, Micha; Parvari, Ruti

2011-01-01

In primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility. The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the β strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD. PMID:21496787

Loss of Centrobin Enables Daughter Centrioles to Form Sensory Cilia in Drosophila.

PubMed

Gottardo, Marco; Pollarolo, Giulia; Llamazares, Salud; Reina, Jose; Riparbelli, Maria G; Callaini, Giuliano; Gonzalez, Cayetano

2015-08-31

Sensory cilia are organelles that convey information to the cell from the extracellular environment. In vertebrates, ciliary dysfunction results in ciliopathies that in humans comprise a wide spectrum of developmental disorders. In Drosophila, sensory cilia are found only in the neurons of type I sensory organs, but ciliary dysfunction also has dramatic consequences in this organism because it impairs the mechanosensory properties of bristles and chaetae and leads to uncoordination, a crippling condition that causes lethality shortly after eclosion. The cilium is defined by the ciliary membrane, a protrusion of the cell membrane that envelops the core structure known as the axoneme, a microtubule array that extends along the cilium from the basal body. In vertebrates, basal body function requires centriolar distal and subdistal appendages and satellites. Because these structures are acquired through centriole maturation, only mother centrioles can serve as basal bodies. Here, we show that although centriole maturity traits are lacking in Drosophila, basal body fate is reserved to mother centrioles in Drosophila type I neurons. Moreover, we show that depletion of the daughter-centriole-specific protein Centrobin (CNB) enables daughter centrioles to dock on the cell membrane and to template an ectopic axoneme that, although structurally defective, protrudes out of the cell and is enveloped by a ciliary membrane. Conversely, basal body capability is inhibited in mother centrioles modified to carry CNB. These results reveal the crucial role of CNB in regulating basal body function in Drosophila ciliated sensory organs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Impaired airway mucociliary function reduces antigen-specific IgA immune response to immunization with a claudin-4-targeting nasal vaccine in mice.

PubMed

Suzuki, Hidehiko; Nagatake, Takahiro; Nasu, Ayaka; Lan, Huangwenxian; Ikegami, Koji; Setou, Mitsutoshi; Hamazaki, Yoko; Kiyono, Hiroshi; Yagi, Kiyohito; Kondoh, Masuo; Kunisawa, Jun

2018-02-13

Vaccine delivery is an essential element for the development of mucosal vaccine, but it remains to be investigated how physical barriers such as mucus and cilia affect vaccine delivery efficacy. Previously, we reported that C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) targeted claudin-4, which is expressed by the epithelium associated with nasopharynx-associated lymphoid tissue (NALT), and could be effective as a nasal vaccine delivery. Mice lacking tubulin tyrosine ligase-like family, member 1 (Ttll1-KO mice) showed mucus accumulation in nasal cavity due to the impaired motility of respiratory cilia. Ttll1-KO mice nasally immunized with C-CPE fused to pneumococcal surface protein A (PspA-C-CPE) showed reduced PspA-specific nasal IgA responses, impaired germinal center formation, and decreased germinal center B-cells and follicular helper T cells in the NALT. Although there was no change in the expression of claudin-4 in the NALT epithelium in Ttll1-KO mice, the epithelium was covered by a dense mucus that prevented the binding of PspA-C-CPE to NALT. However, administration of expectorant N-acetylcysteine removed the mucus and rescued the PspA-specific nasal IgA response. These results show that the accumulation of mucus caused by impaired respiratory cilia function is an interfering factor in the C-CPE-based claudin-4-targeting nasal vaccine.

ZMYND10--Mutation Analysis in Slavic Patients with Primary Ciliary Dyskinesia.

PubMed

Kurkowiak, Małgorzata; Ziętkiewicz, Ewa; Greber, Agnieszka; Voelkel, Katarzyna; Wojda, Alina; Pogorzelski, Andrzej; Witt, Michał

2016-01-01

Primary ciliary dyskinesia (PCD) is a rare recessive disease with a prevalence of 1/10,000; its symptoms are caused by a kinetic dysfunction of motile cilia in the respiratory epithelium, flagella in spermatozoids, and primary cilia in the embryonic node. PCD is genetically heterogeneous: genotyping the already known PCD-related genes explains the genetic basis in 60-65% of the cases, depending on the population. While identification of new genes involved in PCD pathogenesis remains crucial, the search for new, population-specific mutations causative for PCD is equally important. The Slavs remain far less characterized in this respect compared to West European populations, which significantly limits diagnostic capability. The main goal of this study was to characterize the profile of causative genetic defects in one of the PCD-causing genes, ZMYND10, in the cohort of PCD patients of Slavic origin. The study was carried out using biological material from 172 unrelated PCD individuals of Polish origin, with no causative mutation found in nine major PCD genes. While none of the previously described mutations was found using the HRM-based screening, a novel frameshift mutation (c.367delC) in ZMYND10, unique for Slavic PCD population, was found in homozygous state in two unrelated PCD patients. Immunofluorescence analysis confirmed the absence of outer and inner dynein arms from the ciliary axoneme, consistent with the already published ZMYND10-mutated phenotype; cDNA analysis revealed the lack of ZMYND10 mRNA, indicating nonsense-mediated decay of the truncated transcript.

Establishment of left–right asymmetry in vertebrate development: the node in mouse embryos

PubMed Central

Komatsu, Yoshihiro

2014-01-01

Establishment of vertebrate left–right asymmetry is a critical process for normal embryonic development. After the discovery of genes expressed asymmetrically along the left–right axis in chick embryos in the mid 1990s, the molecular mechanisms responsible for left–right patterning in vertebrate embryos have been studied extensively. In this review article, we discuss the mechanisms by which the initial symmetry along the left–right axis is broken in the mouse embryo. We focus on the role of primary cilia and molecular mechanisms of ciliogenesis at the node when symmetry is broken and left–right asymmetry is established. The node is considered a signaling center for early mouse embryonic development, and the results we review here have led to a better understanding of how the node functions and establishes left–right asymmetry. PMID:23771646

Establishment of left-right asymmetry in vertebrate development: the node in mouse embryos.

PubMed

Komatsu, Yoshihiro; Mishina, Yuji

2013-12-01

Establishment of vertebrate left-right asymmetry is a critical process for normal embryonic development. After the discovery of genes expressed asymmetrically along the left-right axis in chick embryos in the mid 1990s, the molecular mechanisms responsible for left-right patterning in vertebrate embryos have been studied extensively. In this review article, we discuss the mechanisms by which the initial symmetry along the left-right axis is broken in the mouse embryo. We focus on the role of primary cilia and molecular mechanisms of ciliogenesis at the node when symmetry is broken and left-right asymmetry is established. The node is considered a signaling center for early mouse embryonic development, and the results we review here have led to a better understanding of how the node functions and establishes left-right asymmetry.

A Conserved Role for Girdin in Basal Body Positioning and Ciliogenesis.

PubMed

Nechipurenko, Inna V; Olivier-Mason, Anique; Kazatskaya, Anna; Kennedy, Julie; McLachlan, Ian G; Heiman, Maxwell G; Blacque, Oliver E; Sengupta, Piali

2016-09-12

Primary cilia are ubiquitous sensory organelles that mediate diverse signaling pathways. Cilia position on the cell surface is determined by the location of the basal body (BB) that templates the cilium. The mechanisms that regulate BB positioning in the context of ciliogenesis are largely unknown. Here we show that the conserved signaling and scaffolding protein Girdin localizes to the proximal regions of centrioles and regulates BB positioning and ciliogenesis in Caenorhabditis elegans sensory neurons and human RPE-1 cells. Girdin depletion alters localization of the intercentriolar linker and ciliary rootlet component rootletin, and rootletin knockdown in RPE-1 cells mimics Girdin-dependent phenotypes. C. elegans Girdin also regulates localization of the apical junction component AJM-1, suggesting that in nematodes Girdin may position BBs via rootletin- and AJM-1-dependent anchoring to the cytoskeleton and plasma membrane, respectively. Together, our results describe a conserved role for Girdin in BB positioning and ciliogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanical Strain Determines Cilia Length, Motility, and Planar Position in the Left-Right Organizer.

PubMed

Chien, Yuan-Hung; Srinivasan, Shyam; Keller, Ray; Kintner, Chris

2018-05-07

The Xenopus left-right organizer (LRO) breaks symmetry along the left-right axis of the early embryo by producing and sensing directed ciliary flow as a patterning cue. To carry out this process, the LRO contains different ciliated cell types that vary in cilia length, whether they are motile or sensory, and how they position their cilia along the anterior-posterior (A-P) planar axis. Here, we show that these different cilia features are specified in the prospective LRO during gastrulation, based on anisotropic mechanical strain that is oriented along the A-P axis, and graded in levels along the medial-lateral axis. Strain instructs ciliated cell differentiation by acting on a mesodermal prepattern present at blastula stages, involving foxj1. We propose that differential strain is a graded, developmental cue, linking the establishment of an A-P planar axis to cilia length, motility, and planar location during formation of the Xenopus LRO. Copyright © 2018 Elsevier Inc. All rights reserved.

High-aspect ratio magnetic nanocomposite polymer cilium

NASA Astrophysics Data System (ADS)

Rahbar, M.; Tseng, H. Y.; Gray, B. L.

2014-03-01

This paper presents a new fabrication technique to achieve ultra high-aspect ratio artificial cilia micro-patterned from flexible highly magnetic rare earth nanoparticle-doped polymers. We have developed a simple, inexpensive and scalable fabrication method to create cilia structures that can be actuated by miniature electromagnets, that are suitable to be used for lab-on-a chip (LOC) and micro-total-analysis-system (μ-TAS) applications such as mixers and flow-control elements. The magnetic cilia are fabricated and magnetically polarized directly in microfluidic channels or reaction chambers, allowing for easy integration with complex microfluidic systems. These cilia structures can be combined on a single chip with other microfluidic components employing the same permanently magnetic nano-composite polymer (MNCP), such as valves or pumps. Rare earth permanent magnetic powder, (Nd0.7Ce0.3)10.5Fe83.9B5.6, is used to dope polydimethylsiloxane (PDMS), resulting in a highly flexible M-NCP of much higher magnetization and remanence [1] than ferromagnetic polymers typically employed in magnetic microfluidics. Sacrificial poly(ethylene-glycol) (PEG) is used to mold the highly magnetic polymer into ultra high-aspect ratio artificial cilia. Cilia structures with aspect ratio exceeding 8:0.13 can be easily fabricated using this technique and are actuated using miniature electromagnets to achieve a high range of motion/vibration.

Primary Ciliary Dyskinesia.

PubMed

Knowles, Michael R; Zariwala, Maimoona; Leigh, Margaret

2016-09-01

Primary ciliary dyskinesia (PCD) is a recessive genetically heterogeneous disorder of motile cilia with chronic otosinopulmonary disease and organ laterality defects in ∼50% of cases. The prevalence of PCD is difficult to determine. Recent diagnostic advances through measurement of nasal nitric oxide and genetic testing has allowed rigorous diagnoses and determination of a robust clinical phenotype, which includes neonatal respiratory distress, daily nasal congestion, and wet cough starting early in life, along with organ laterality defects. There is early onset of lung disease in PCD with abnormal airflow mechanics and radiographic abnormalities detected in infancy and early childhood. Copyright © 2016 Elsevier Inc. All rights reserved.

Immobilization antibodies of tiger puffer Takifugu rubripes induced by i.p. injection against monogenean Heterobothrium okamotoi oncomiracidia do not prevent the infection.

PubMed

Umeda, N; Hatanaka, A; Hirazawa, N

2007-06-01

We examined whether infection by the monogenean Heterobothrium okamotoi induces production of specific antibodies against oncomiracidia and their cilia, larvae on the gills, and adults on the branchial cavity wall of tiger puffer Takifugu rubripes. We also investigated whether specific antibody production participates in acquired protection against H. okamotoi. Sera from persistently infected fish immobilized H. okamotoi oncomiracidia 89 days after exposure and antibody levels (measured by enzyme-linked immunosorbent assays) in the sera against oncomiracidia and their cilia increased compared with sera from control (naïve) fish. Antibody levels in these sera against the larvae and adult stages did not increase. The number of H. okamotoi on persistently infected fish was significantly lower than for control fish (P<0.05) when persistently infected fish and control fish were exposed to oncomiracidia in the same tank. Thus tiger puffer produced specific antibodies against oncomiracidia and their cilia, and acquired partial protection against H. okamotoi. Intraperitoneal injection of proteins of sonicated oncomiracidia or their cilia with an adjuvant also produced oncomiracidium agglutination antibodies in sera from tiger puffer; the antibody levels in these sera against oncomiracidia and their cilia increased compared with sera from control fish (injection of BSA with an adjuvant) at 14, 44, and 75 days after the booster immunization. However, in a parasite challenge at 54-58 days after the booster immunization, the infection levels of fish immunized with parasites of sonicated oncomiracidia or their cilia were the same as the control fish. Western blot showed that sera from persistently infected fish and fish immunized with sonicated oncomiracidia or their cilia recognized similar antigenic bands, suggesting that tiger puffer tends to react against these antigens compared with other antigens. These results indicated that specific antibodies against these cilia and oncomiracidia induced by i.p. injection do not prevent H. okamotoi infection.

Discrete cilia modelling with singularity distributions: application to the embryonic node and the airway surface liquid.

PubMed

Smith, D J; Gaffney, E A; Blake, J R

2007-07-01

We discuss in detail techniques for modelling flows due to finite and infinite arrays of beating cilia. An efficient technique, based on concepts from previous 'singularity models' is described, that is accurate in both near and far-fields. Cilia are modelled as curved slender ellipsoidal bodies by distributing Stokeslet and potential source dipole singularities along their centrelines, leading to an integral equation that can be solved using a simple and efficient discretisation. The computed velocity on the cilium surface is found to compare favourably with the boundary condition. We then present results for two topics of current interest in biology. 1) We present the first theoretical results showing the mechanism by which rotating embryonic nodal cilia produce a leftward flow by a 'posterior tilt,' and track particle motion in an array of three simulated nodal cilia. We find that, contrary to recent suggestions, there is no continuous layer of negative fluid transport close to the ciliated boundary. The mean leftward particle transport is found to be just over 1 mum/s, within experimentally measured ranges. We also discuss the accuracy of models that represent the action of cilia by steady rotlet arrays, in particular, confirming the importance of image systems in the boundary in establishing the far-field fluid transport. Future modelling may lead to understanding of the mechanisms by which morphogen gradients or mechanosensing cilia convert a directional flow to asymmetric gene expression. 2) We develop a more complex and detailed model of flow patterns in the periciliary layer of the airway surface liquid. Our results confirm that shear flow of the mucous layer drives a significant volume of periciliary liquid in the direction of mucus transport even during the recovery stroke of the cilia. Finally, we discuss the advantages and disadvantages of the singularity technique and outline future theoretical and experimental developments required to apply this technique to various other biological problems, particularly in the reproductive system.

3D structure of eukaryotic flagella/cilia by cryo-electron tomography.

PubMed

Ishikawa, Takashi

2013-01-01

Flagella/cilia are motile organelles with more than 400 proteins. To understand the mechanism of such complex systems, we need methods to describe molecular arrange-ments and conformations three-dimensionally in vivo. Cryo-electron tomography enabled us such a 3D structural analysis. Our group has been working on 3D structure of flagella/cilia using this method and revealed highly ordered and beautifully organized molecular arrangement. 3D structure gave us insights into the mechanism to gener-ate bending motion with well defined waveforms. In this review, I summarize our recent structural studies on fla-gella/cilia by cryo-electron tomography, mainly focusing on dynein microtubule-based ATPase motor proteins and the radial spoke, a regulatory protein complex.

3D structure of eukaryotic flagella/cilia by cryo-electron tomography

PubMed Central

Ishikawa, Takashi

2013-01-01

Flagella/cilia are motile organelles with more than 400 proteins. To understand the mechanism of such complex systems, we need methods to describe molecular arrange-ments and conformations three-dimensionally in vivo. Cryo-electron tomography enabled us such a 3D structural analysis. Our group has been working on 3D structure of flagella/cilia using this method and revealed highly ordered and beautifully organized molecular arrangement. 3D structure gave us insights into the mechanism to gener-ate bending motion with well defined waveforms. In this review, I summarize our recent structural studies on fla-gella/cilia by cryo-electron tomography, mainly focusing on dynein microtubule-based ATPase motor proteins and the radial spoke, a regulatory protein complex. PMID:27493552

Drosophila Ana1 is required for centrosome assembly and centriole elongation

PubMed Central

Saurya, Saroj; Roque, Hélio; Novak, Zsofia A.; Wainman, Alan; Aydogan, Mustafa G.; Volanakis, Adam; Sieber, Boris; Pinto, David Miguel Susano

2016-01-01

ABSTRACT Centrioles organise centrosomes and cilia, and these organelles have an important role in many cell processes. In flies, the centriole protein Ana1 is required for the assembly of functional centrosomes and cilia. It has recently been shown that Cep135 (also known as Bld10) initially recruits Ana1 to newly formed centrioles, and that Ana1 then recruits Asl (known as Cep152 in mammals) to promote the conversion of these centrioles into centrosomes. Here, we show that ana1 mutants lack detectable centrosomes in vivo, that Ana1 is irreversibly incorporated into centrioles during their assembly and appears to play a more important role in maintaining Asl at centrioles than in initially recruiting Asl to centrioles. Unexpectedly, we also find that Ana1 promotes centriole elongation in a dose-dependent manner: centrioles are shorter when Ana1 dosage is reduced and are longer when Ana1 is overexpressed. This latter function of Ana1 appears to be distinct from its role in centrosome and cilium function, as a GFP–Ana1 fusion lacking the N-terminal 639 amino acids of the protein can support centrosome assembly and cilium function but cannot promote centriole over-elongation when overexpressed. PMID:27206860

Flow generation by the corona ciliata in Chaetognatha - quantification and implications for current functional hypotheses.

PubMed

Bleich, Steffen; Müller, Carsten H G; Graf, Gerhard; Hanke, Wolf

2017-12-01

The corona ciliata of Chaetognatha (arrow worms) is a circular or elliptical groove lined by a rim from which multiple lines of cilia emanate, located dorsally on the head and/or trunk. Mechanoreception, chemosensation, excretion, respiration, and support of reproduction have been suggested to be its main functions. Here we provide the first experimental evidence that the cilia produce significant water flow, and the first visualisation and quantification of this flow. In Spadella cephaloptera, water is accelerated toward the corona ciliata from dorsal and anterior of the body in a funnel-shaped pattern, and expelled laterally and caudally from the corona, with part of the water being recirculated. Maximal flow speeds were approximately 140μms -1 in adult specimens. Volumetric flow rate was Q=0.0026μls -1 . The funnel-shaped directional flow can possibly enable directional chemosensation. The flow measurements demonstrate that the corona ciliata is well suited as a multifunctional organ. Copyright © 2017 Elsevier GmbH. All rights reserved.

["Light" epithelial cells of swine and bovine oviducts].

PubMed

Suuroia, T; Aunapuu, M; Arend, A; Sépp, E

2002-01-01

The ultrastructure of oviduct epithelium of clinically healthy cows and 15 sows was investigated using scanning and transmission electron microscopy. In all parts of the oviduct, ciliated and non-ciliated epithelial cells are present, but their number varies in both the investigated animals in different regions of the oviduct, depending on the phase of the estrous cycle. In addition to ciliated cells with numerous cilia on their luminal surface, so-called pale ciliary cells were found in all parts of the oviduct of cows and sows. The cytoplasm of these cells is electron-lucent, their luminal surface carries few cilia and short microvilli. The apical cytoplasm contains species specific secretory granules, which means that these cells have features characteristic of both secretory and ciliated cells. It is suggested that the pale ciliated and non-ciliated secretory cells are functional stages of the same tubar epithelium cell, and that the transformation between these two cell types is regulated by functional requirements of the organ in different phases of the estrous cycle.

Hippi is essential for node cilia assembly and Sonic hedgehog signaling

PubMed Central

Houde, Caroline; Dickinson, Robin J.; Houtzager, Vicky M.; Cullum, Rebecca; Montpetit, Rachel; Metzler, Martina; Simpson, Elizabeth M.; Roy, Sophie; Hayden, Michael R.; Hoodless, Pamela A.; Nicholson, Donald W.

2016-01-01

Hippi functions as an adapter protein that mediates pro-apoptotic signaling from poly-glutamine-expanded huntingtin, an established cause of Huntington disease, to the extrinsic cell death pathway. To explore other functions of Hippi we generated Hippi knock-out mice. This deletion causes randomization of the embryo turning process and heart looping, which are hallmarks of defective left–right (LR) axis patterning. We report that motile monocilia normally present at the surface of the embryonic node, and proposed to initiate the break in LR symmetry, are absent on Hippi−/− embryos. Furthermore, defects in central nervous system development are observed. The Sonic hedgehog (Shh) pathway is downregulated in the neural tube in the absence of Hippi, which results in failure to establish ventral neural cell fate. Together, these findings demonstrate a dual role for Hippi in cilia assembly and Shh signaling during development, in addition to its proposed role in apoptosis signal transduction in the adult brain under pathogenically stressful conditions. PMID:17027958

Quantification and visualization of injury and regeneration to the ciliated epithelium using quantitative flow imaging and speckle variance optical coherence tomography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Gamm, Ute A.; Huang, Brendan K.; Mis, Emily K.; Khokha, Mustafa K.; Choma, Michael A.

2017-04-01

Mucociliary flow is an important defense mechanism in the lung to remove inhaled pathogens and pollutants. A disruption of ciliary flow can lead to respiratory infections. Even though patients in the intensive care unit (ICU) either have or are very susceptible to respiratory infections, mucociliary flow is not well understood in the ICU setting. We recently demonstrated that hyperoxia, a consequence of administering supplemental oxygen to a patient in respiratory failure, can lead to a significant reduction of cilia-driven fluid flow in mouse trachea. There are other factors that are relevant to ICU medicine that can damage the ciliated tracheal epithelium, including inhalation injury and endotracheal tube placement. In this study we use two animal models, Xenopus embryo and ex vivo mouse trachea, to analyze flow defects in the injured ciliated epithelium. Injury is generated either mechanically with a scalpel or chemically by calcium chloride (CaCl2) shock, which efficiently but reversibly deciliates the embryo skin. In this study we used optical coherence tomography (OCT) and particle tracking velocimetry (PTV) to quantify cilia driven fluid flow over the surface of the Xenopus embryo. We additionally visualized damage to the ciliated epithelium by capturing 3D speckle variance images that highlight beating cilia. Mechanical injury disrupted cilia-driven fluid flow over the injured site, which led to a reduction in cilia-driven fluid flow over the whole surface of the embryo (n=7). The calcium chloride shock protocol proved to be highly effective in deciliating embryos (n=6). 3D speckle variance images visualized a loss of cilia and cilia-driven flow was halted immediately after application. We also applied CaCl2-shock to cultured ex vivo mouse trachea (n=8) and found, similarly to effects in Xenopus embryo, an extensive loss of cilia with resulting cessation of flow. We investigated the regeneration of the ciliated epithelium after an 8 day incubation period, and found that cilia had regrown and flow was completely restored. In conclusion, OCT is a valuable tool to visualize injury of the ciliated epithelium and to quantify reduction of generated flow. This method allows for systematic investigation of focal and diffuse injury of the ciliated epithelium and the assessment of mechanisms to compensate for loss of flow.

Recent advances in primary ciliary dyskinesia genetics

PubMed Central

Kurkowiak, Małgorzata; Ziętkiewicz, Ewa; Witt, Michał

2015-01-01

Primary ciliary dyskinesia (PCD) is a rare genetically heterogeneous disorder caused by the abnormal structure and/or function of motile cilia. The PCD diagnosis is challenging and requires a well-described clinical phenotype combined with the identification of abnormalities in ciliary ultrastructure and/or beating pattern as well as the recognition of genetic cause of the disease. Regarding the pace of identification of PCD-related genes, a rapid acceleration during the last 2–3 years is notable. This is the result of new technologies, such as whole-exome sequencing, that have been recently applied in genetic research. To date, PCD-causative mutations in 29 genes are known and the number of causative genes is bound to rise. Even though the genetic causes of approximately one-third of PCD cases still remain to be found, the current knowledge can already be used to create new, accurate genetic tests for PCD that can accelerate the correct diagnosis and reduce the proportion of unexplained cases. This review aims to present the latest data on the relations between ciliary structure aberrations and their genetic basis. PMID:25351953

Cilia in the head of hornets: form and function.

PubMed

Ishay, Jacob S; Plotkin, Marian; Ermakov, Natalya; Jongebloed, Willem L; Kalicharan, Dharamdajal; Bergman, David J

2005-06-01

In the head of the Oriental hornet, beneath the cuticle, there are plaques of hair cells. These are distributed throughout the upper front part of the head; to wit: in the region of the vertex (i.e., around and behind the ocelli), in the genae around and behind the compound eyes (the ommatidia), and in the region of the forehead or frons. These hair cells are arranged with their thin whip-like part (i.e., cilia) directed outward and morphologically fall into three distinct groups: type (a) thin elongated cilia connected to each other alongside by side-links; type (b) thin elongated cilia of which two or more interconnect at their distal ends via a delicate nerve fiber bearing a knob at its center; and type (c) shorter and thicker cilia that roughly resemble a triangular thorn and are also interconnected by a thin thread, which, however, bears a ball rather then a knob at its center. The knob in the one case and the ball in the other vary in their diameter, but in both instances the interconnecting elements, be they nerve fibers or threads, are seemingly multidirectional. Beneath the frons, in the region of the coronal suture, the hair cells (cilial plaques) are inwardly directed and bear a large trachea at their center. Presumably, the "weighted" cilial cells that are directed toward the exterior of the body aid the hornet in navigation and gravity determination whereas the inwardly directed ciliary cells may possibly serve in acoustic communication. Another element worthy of mention within the hair cells are yellow granules (yg). These yg's originate from the whip-like portion of the ciliary cells that are distributed beneath the frons plate, and also in the yellow stripes of the gastral cuticle. Conceivably, these yellow granules, in both cases, may play a role in the absorption and storage of solar energy. In summary, ciliary structures are involved in the hornet in gravity sensing, in acoustical communication and in light sensing, i.e., with some similarity with what happens in vertebrates in the inner ear and in the photoreceptor. Copyright (c) 2005 Wiley-Liss, Inc.

Bug22p, a Conserved Centrosomal/Ciliary Protein Also Present in Higher Plants, Is Required for an Effective Ciliary Stroke in Paramecium ▿ †

PubMed Central

Laligné, C.; Klotz, C.; Garreau de Loubresse, N.; Lemullois, M.; Hori, M.; Laurent, F. X.; Papon, J. F.; Louis, B.; Cohen, J.; Koll, F.

2010-01-01

Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate. PMID:20118210

Bug22p, a conserved centrosomal/ciliary protein also present in higher plants, is required for an effective ciliary stroke in Paramecium.

PubMed

Laligné, C; Klotz, C; de Loubresse, N Garreau; Lemullois, M; Hori, M; Laurent, F X; Papon, J F; Louis, B; Cohen, J; Koll, F

2010-04-01

Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate.

Analytical approach to entropy generation and heat transfer in CNT-nanofluid dynamics through a ciliated porous medium

NASA Astrophysics Data System (ADS)

Akbar, Noreen Sher; Shoaib, M.; Tripathi, Dharmendra; Bhushan, Shashi; Bég, O. Anwar

2018-04-01

The transportation of biological and industrial nanofluids by natural propulsion like cilia movement and self-generated contraction-relaxation of flexible walls has significant applications in numerous emerging technologies. Inspired by multi-disciplinary progress and innovation in this direction, a thermo-fluid mechanical model is proposed to study the entropy generation and convective heat transfer of nanofluids fabricated by the dispersion of single-wall carbon nanotubes (SWCNT) nanoparticles in water as the base fluid. The regime studied comprises heat transfer and steady, viscous, incompressible flow, induced by metachronal wave propulsion due to beating cilia, through a cylindrical tube containing a sparse (i.e., high permeability) homogenous porous medium. The flow is of the creeping type and is restricted under the low Reynolds number and long wavelength approximations. Slip effects at the wall are incorporated and the generalized Darcy drag-force model is utilized to mimic porous media effects. Cilia boundary conditions for velocity components are employed to determine analytical solutions to the resulting non-dimensionalized boundary value problem. The influence of pertinent physical parameters on temperature, axial velocity, pressure rise and pressure gradient, entropy generation function, Bejan number and stream-line distributions are computed numerically. A comparative study between SWCNT-nanofluids and pure water is also computed. The computations demonstrate that axial flow is accelerated with increasing slip parameter and Darcy number and is greater for SWCNT-nanofluids than for pure water. Furthermore the size of the bolus for SWCNT-nanofluids is larger than that of the pure water. The study is applicable in designing and fabricating nanoscale and microfluidics devices, artificial cilia and biomimetic micro-pumps.

Analytical approach to entropy generation and heat transfer in CNT-nanofluid dynamics through a ciliated porous medium

NASA Astrophysics Data System (ADS)

Akbar, Noreen Sher; Shoaib, M.; Tripathi, Dharmendra; Bhushan, Shashi; Bég, O. Anwar

2018-03-01

The transportation of biological and industrial nanofluids by natural propulsion like cilia movement and self-generated contraction-relaxation of flexible walls has significant applications in numerous emerging technologies. Inspired by multi-disciplinary progress and innovation in this direction, a thermo-fluid mechanical model is proposed to study the entropy generation and convective heat transfer of nanofluids fabricated by the dispersion of single-wall carbon nanotubes (SWCNT) nanoparticles in water as the base fluid. The regime studied comprises heat transfer and steady, viscous, incompressible flow, induced by metachronal wave propulsion due to beating cilia, through a cylindrical tube containing a sparse (i.e., high permeability) homogenous porous medium. The flow is of the creeping type and is restricted under the low Reynolds number and long wavelength approximations. Slip effects at the wall are incorporated and the generalized Darcy drag-force model is utilized to mimic porous media effects. Cilia boundary conditions for velocity components are employed to determine analytical solutions to the resulting non-dimensionalized boundary value problem. The influence of pertinent physical parameters on temperature, axial velocity, pressure rise and pressure gradient, entropy generation function, Bejan number and stream-line distributions are computed numerically. A comparative study between SWCNT-nanofluids and pure water is also computed. The computations demonstrate that axial flow is accelerated with increasing slip parameter and Darcy number and is greater for SWCNT-nanofluids than for pure water. Furthermore the size of the bolus for SWCNT-nanofluids is larger than that of the pure water. The study is applicable in designing and fabricating nanoscale and microfluidics devices, artificial cilia and biomimetic micro-pumps.

ICK is essential for cell type-specific ciliogenesis and the regulation of ciliary transport.

PubMed

Chaya, Taro; Omori, Yoshihiro; Kuwahara, Ryusuke; Furukawa, Takahisa

2014-06-02

Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport; however, the underlying regulatory mechanism is unclear. Here, we identified ICK localization at the tip of cilia as a regulator of ciliary transport. In ICK-deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK-deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT-A, IFT-B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT-B, but not IFT-A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia. © 2014 The Authors.

ICK is essential for cell type-specific ciliogenesis and the regulation of ciliary transport

PubMed Central

Chaya, Taro; Omori, Yoshihiro; Kuwahara, Ryusuke; Furukawa, Takahisa

2014-01-01

Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport; however, the underlying regulatory mechanism is unclear. Here, we identified ICK localization at the tip of cilia as a regulator of ciliary transport. In ICK-deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK-deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT-A, IFT-B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT-B, but not IFT-A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia. PMID:24797473

Microtubules Enable the Planar Cell Polarity of Airway Cilia

PubMed Central

Vladar, Eszter K.; Bayly, Roy D.; Sangoram, Ashvin; Scott, Matthew P.; Axelrod, Jeffrey D.

2012-01-01

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. PMID:23122850

Metachronal waves in epithelium cilia to transport bronchial mucus in airways

NASA Astrophysics Data System (ADS)

Favier, Julien; Sylvain, Chateau; D'Ortona, Umberto; Poncet, Sébastien

2017-11-01

Metachronal waves of beating cilia are an efficient mechanism to transport mucus in human airways. The numerical results we will present will shed new light on the understanding of chronic respiratory diseases, such as Asthma of COPD. A coupled lattice Boltzmann - Immersed Boundary is used to simulate the multiphase environment in which the cilia are immersed: a periciliary layer and the mucus layer. A purely hydrodynamical feedback of the fluids is taken into account, and a coupling parameter α is introduced, allowing the tuning of both the direction of the wave propagation, and the strength of the fluid feedback. The cilia, initially set in a random state, quickly synchronize with their immediate neighbors giving birth to metachronal waves. A comparative study of both antipleptic and sympleptic waves is performed by imposing the metachrony. Antiplectic waves are found to be the most efficient to transport and mix fluids compared to other random or synchronised cilia motions. The numerical results will be discussed and compared to experimental and clinical results obtained by collaborators, to progress on the understanding of the inner mechanisms of chronic respiratory diseases.

The V-ATPase accessory protein Atp6ap1b mediates dorsal forerunner cell proliferation and left-right asymmetry in zebrafish.

PubMed

Gokey, Jason J; Dasgupta, Agnik; Amack, Jeffrey D

2015-11-01

Asymmetric fluid flows generated by motile cilia in a transient 'organ of asymmetry' are involved in establishing the left-right (LR) body axis during embryonic development. The vacuolar-type H(+)-ATPase (V-ATPase) proton pump has been identified as an early factor in the LR pathway that functions prior to cilia, but the role(s) for V-ATPase activity are not fully understood. In the zebrafish embryo, the V-ATPase accessory protein Atp6ap1b is maternally supplied and expressed in dorsal forerunner cells (DFCs) that give rise to the ciliated organ of asymmetry called Kupffer's vesicle (KV). V-ATPase accessory proteins modulate V-ATPase activity, but little is known about their functions in development. We investigated Atp6ap1b and V-ATPase in KV development using morpholinos, mutants and pharmacological inhibitors. Depletion of both maternal and zygotic atp6ap1b expression reduced KV organ size, altered cilia length and disrupted LR patterning of the embryo. Defects in other ciliated structures-neuromasts and olfactory placodes-suggested a broad role for Atp6ap1b during development of ciliated organs. V-ATPase inhibitor treatments reduced KV size and identified a window of development in which V-ATPase activity is required for proper LR asymmetry. Interfering with Atp6ap1b or V-ATPase function reduced the rate of DFC proliferation, which resulted in fewer ciliated cells incorporating into the KV organ. Analyses of pH and subcellular V-ATPase localizations suggested Atp6ap1b functions to localize the V-ATPase to the plasma membrane where it regulates proton flux and cytoplasmic pH. These results uncover a new role for the V-ATPase accessory protein Atp6ap1b in early development to maintain the proliferation rate of precursor cells needed to construct a ciliated KV organ capable of generating LR asymmetry. Copyright © 2015 Elsevier Inc. All rights reserved.

The V-ATPase accessory protein Atp6ap1b mediates dorsal forerunner cell proliferation and left-right asymmetry in zebrafish

PubMed Central

Gokey, Jason J.; Dasgupta, Agnik; Amack, Jeffrey D.

2015-01-01

Asymmetric fluid flows generated by motile cilia in a transient ‘organ of asymmetry’ are involved in establishing the left-right (LR) body axis during embryonic development. The vacuolar-type H+-ATPase (V-ATPase) proton pump has been identified as an early factor in the LR pathway that functions prior to cilia, but the role(s) for V-ATPase activity are not fully understood. In the zebrafish embryo, the V-ATPase accessory protein Atp6ap1b is maternally supplied and expressed in dorsal forerunner cells (DFCs) that give rise to the ciliated organ of asymmetry called Kupffer’s vesicle (KV). V-ATPase accessory proteins modulate V-ATPase activity, but little is known about their functions in development. We investigated Atp6ap1b and V-ATPase in KV development using morpholinos, mutants and pharmacological inhibitors. Depletion of both maternal and zygotic atp6ap1b expression reduced KV organ size, altered cilia length and disrupted LR patterning of the embryo. Defects in other ciliated structures—neuromasts and olfactory placodes—suggested a broad role for Atp6ap1b during development of ciliated organs. V-ATPase inhibitor treatments reduced KV size and identified a window of development in which V-ATPase activity is required for proper LR asymmetry. Interfering with Atp6ap1b or V-ATPase function reduced the rate of DFC proliferation, which resulted in fewer ciliated cells incorporating into the KV organ. Analyses of pH and subcellular V-ATPase localizations suggested Atp6ap1b functions to localize the V-ATPase to the plasma membrane where it regulates proton flux and cytoplasmic pH. These results uncover a new role for the V-ATPase accessory protein Atp6ap1b in early development to maintain the proliferation rate of precursor cells needed to construct a ciliated KV organ capable of generating LR asymmetry. PMID:26254189

Double life of centrioles: CP110 in the spotlight.

PubMed

Bettencourt-Dias, Mónica; Carvalho-Santos, Zita

2008-01-01

Centrioles lead an important double life: they can give rise to the centrosome or convert to basal bodies and template cilia. Little is known about the control of centriole fate. Spektor and colleagues have now identified a centriolar complex, composed of CP110 and CEP97, which inhibits centriole to basal body conversion, preventing cilia formation. This work paves the way to understanding centriole and cilia biogenesis, which are two processes misregulated in human diseases, such as cancer and polycystic kidney disease.

[A case of Kartagener's syndrome].

PubMed

Ishiga, Takeshi; Tanigawa, Motoaki; Ichioka, Maresuke; Saito, Kimimasa

2005-03-01

This case describes a 57-year-old woman in whom situs inversus had been noted at her birth. She had bronchial asthma and bilateral sinusitis during her childhood. She married and experienced childbirth. In December 2003, she was admitted to our Division complaining of wheezing, expectoration and dyspnea on effort. Bronciectasis was visualized on chest X-ray and CT. Electron microscopic examination of the nasal cavity epithelium and bronchial epithelial cilia revealed a deficit of bilateral dynein arms. These findings, helped establish a diagnosis of Kartagener's syndrome, which is characterized by primary ciliary dyskinesia. The restrictive and obstructive pulmonary dysfunction with increase of residual volume in the lung function tests and diffuse centrilobular small nodules with hyperinflation on chest CT were consistent with the findings of diffuse panbronchilitis (DPB) and suggested extended obliterative peripheral airway disease. Clarithromycin which is highly effective for DPB failed to prevent the aggravation of airway infection, arousing the concern about the progression into chronic respiratory failure.

An essential role of intestinal cell kinase in lung development is linked to the perinatal lethality of human ECO syndrome

PubMed Central

Tong, Yixin; Park, So Hyun; Wu, Di; Xu, Wenhao; Guillot, Stacey J.; Jin, Li; Li, Xudong; Wang, Yalin; Lin, Chyuan-Sheng; Fu, Zheng

2017-01-01

Human endocrine-cerebro-osteodysplasia (ECO) syndrome, caused by the loss-of-function mutation R272Q in the ICK (intestinal cell kinase) gene, is a neonatal-lethal developmental disorder. To elucidate the molecular basis of ECO syndrome, we constructed an Ick R272Q knock-in mouse model that recapitulates ECO pathological phenotypes. Newborns bearing Ick R272Q homozygous mutations die at birth due to respiratory distress. Ick mutant lungs exhibit not only impaired branching morphogenesis associated with reduced mesenchymal proliferation, but also significant airspace deficiency in primitive alveoli concomitant with abnormal interstitial mesenchymal differentiation. ICK dysfunction induces elongated primary cilia and perturbs ciliary Hedgehog signaling and autophagy during lung sacculation. Our study identifies an essential role for ICK in lung development and advances the mechanistic understanding of ECO syndrome. PMID:28380258

An essential role of intestinal cell kinase in lung development is linked to the perinatal lethality of human ECO syndrome.

PubMed

Tong, Yixin; Park, So Hyun; Wu, Di; Xu, Wenhao; Guillot, Stacey J; Jin, Li; Li, Xudong; Wang, Yalin; Lin, Chyuan-Sheng; Fu, Zheng

2017-05-01

Human endocrine-cerebro-osteodysplasia (ECO) syndrome, caused by the loss-of-function mutation R272Q in the intestinal cell kinase (ICK) gene, is a neonatal-lethal developmental disorder. To elucidate the molecular basis of ECO syndrome, we constructed an Ick R272Q knock-in mouse model that recapitulates ECO pathological phenotypes. Newborns bearing Ick R272Q homozygous mutations die at birth due to respiratory distress. Ick mutant lungs exhibit not only impaired branching morphogenesis associated with reduced mesenchymal proliferation but also significant airspace deficiency in primitive alveoli concomitant with abnormal interstitial mesenchymal differentiation. ICK dysfunction induces elongated primary cilia and perturbs ciliary Hedgehog signaling and autophagy during lung sacculation. Our study identifies an essential role for ICK in lung development and advances the mechanistic understanding of ECO syndrome. © 2017 Federation of European Biochemical Societies.

Left-Right Asymmetry: Myosin 1D at the Center.

PubMed

Yuan, Shiaulou; Brueckner, Martina

2018-05-07

While a ciliated organizer generates vertebrate left-right asymmetry, most invertebrates lack an organizer and instead utilize a myosin-based mechanism. A recent study now reveals that this myosin mechanism is conserved in the vertebrate organizer and functions to regulate cilia. Copyright © 2018 Elsevier Ltd. All rights reserved.

Primary ciliary dyskinesia caused by homozygous mutation in DNAL1, encoding dynein light chain 1.

PubMed

Mazor, Masha; Alkrinawi, Soliman; Chalifa-Caspi, Vered; Manor, Esther; Sheffield, Val C; Aviram, Micha; Parvari, Ruti

2011-05-13

In primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility. The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the β strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Genetics Home Reference: Bardet-Biedl syndrome

MedlinePlus

... signaling pathways. Cilia are also necessary for the perception of sensory input (such as sight, hearing, and ... during development and lead to abnormalities of sensory perception. Researchers believe that defective cilia are responsible for ...

Presence of Ca2+-dependent K+ channels in chemosensory cilia support a role in odor transduction.

PubMed

Delgado, Ricardo; Saavedra, M Veronica; Schmachtenberg, Oliver; Sierralta, Jimena; Bacigalupo, Juan

2003-09-01

Olfactory receptor neurons (ORNs) respond to odorants with changes in the action potential firing rate. Excitatory responses, consisting of firing increases, are mediated by a cyclic AMP cascade that leads to the activation of cationic nonselective cyclic nucleotide-gated (CNG) channels and Ca2+-dependent Cl- (ClCa) channels. This process takes place in the olfactory cilia, where all protein components of this cascade are confined. ORNs from various vertebrate species have also been shown to generate inhibitory odor responses, expressed as decreases in action potential discharges. Odor inhibition appears to rely on Ca2+-dependent K+ (KCa) channels, but the underlying transduction mechanism remains unknown. If these channels are involved in odor transduction, they are expected to be present in the olfactory cilia. We found that a specific antibody against a large conductance KCa recognized a protein of approximately 116 kDa in Western blots of purified rat olfactory ciliary membranes. Moreover, the antibody labeled ORN cilia in isolated ORNs from rat and toad (Caudiverbera caudiverbera). In addition, single-channel recordings from inside-out membrane patches excised from toad chemosensory cilia showed the presence of 4 different types of KCa channels, with unitary conductances of 210, 60, 12, and 29 and 60 pS, high K+-selectivity, and Ca2+ sensitivities in the low micromolar range. Our work demonstrates the presence of K+ channels in the ORN cilia and supports their participation in odor transduction.

Contrary effects of octopamine receptor ligands on behavioral and neuronal changes in locomotion of lymnaea.

PubMed

Miyamae, Yurie; Komuro, Mami; Murata, Aya; Aono, Kanako; Nishikata, Kaori; Kanazawa, Akira; Fujito, Yutaka; Komatsu, Takumi; Ito, Daisuke; Abe, Takashi; Nagayama, Masafumi; Uchida, Tsutomu; Gohara, Kazutoshi; Murakami, Jun; Kawai, Ryo; Hatakeyama, Dai; Lukowiak, Ken; Ito, Etsuro

2010-02-01

The pond snail Lymnaea stagnalis moves along the sides and bottom of an aquarium, but it can also glide upside down on its back below the water's surface. We have termed these two forms of locomotion "standard locomotion" and "upside-down gliding," respectively. Previous studies showed that standard locomotion is produced by both cilia activity on the foot and peristaltic contraction of the foot muscles, whereas upside-down gliding is mainly caused by cilia activity. The pedal A neurons are thought to receive excitatory octopaminergic input, which ultimately results in increased cilia beating. However, the relationship between locomotory speed and the responses of these neurons to octopamine is not known. We thus examined the effects of both an agonist and an antagonist of octopamine receptors on locomotory speed and the firing rate of the pedal A neurons. We also examined, at the electron and light-microscopic levels, whether structural changes occur in cilia following the application of either an agonist or an antagonist of octopamine receptors to the central nervous system (CNS). We found that the application of an octopamine antagonist to the CNS increased the speed of both forms of locomotion, whereas application of octopamine increased only the firing rate of the pedal A neurons. Microscopic examination of the cilia proved that there were no changes in their morphology after application of octopamine ligands. These data suggest that there is an unidentified octopaminergic neuronal network in the CNS whose activation reduces cilia movement and thus locomotory speed.

Ciliary behavior of a negatively phototactic Chlamydomonas reinhardtii.

PubMed

Josef, Keith; Saranak, Jureepan; Foster, Kenneth W

2005-06-01

With an instrument that can record the motion of both cilia of the unicellular alga Chlamydomonas reinhardtii for many hours, the behavioral differences of its two cilia have been studied to determine their specific role in phototaxis. The organism was held on a fixed micropipette with the plane of ciliary beating rotated into the imaging plane of a quadrant photodetector. The responses to square-wave light patterns of a wide range of temporal frequencies were used to characterize the responses of each cilium. Eighty-one cells were examined showing an unexpectedly diverse range of responses. Plausible common signals for the linear and nonlinear signals from the cell body are suggested. Three independent ciliary measures--the beat frequency, stroke velocity, and phasing of the two cilia--have been identified. The cell body communicates to the cilia the direction of phototaxis the cell desires to go, the absolute light intensity, and the appropriate graded transient response for tracking the light source. The complexity revealed by each measure of the ciliary response indicates many independent variables are involved in the net phototactic response. In spite of their morphological similarity, the two cilia of Chlamydomonas respond uniquely. Probably the signals from the cell body fan out to independent pathways in the cilia. Each cilium modifies the input in its own way. The change in the pattern of the effective and recovery strokes of each cilium associated with negative phototaxis has been demonstrated and its involvement in phototactic turning is described. Copyright (c) 2005 Wiley-Liss, Inc.

Cilia induced cerebrospinal fluid flow in the third ventricle of brain

NASA Astrophysics Data System (ADS)

Wang, Yong; Westendorf, Christian; Faubel, Regina; Eichele, Gregor; Bodenschatz, Eberhard

2016-11-01

Cerebrospinal fluid (CSF) conveys many physiologically important signaling factors through the ventricles of the mammalian brain. The walls of the ventricles are covered with motile cilia that were thought to generate a laminar flow purely following the curvature of walls. However, we recently discovered that cilia of the ventral third ventricle (v3V) generate a complex flow network along the wall, leading to subdivision of the v3V. The contribution of such cilia induced flow to the overall three dimensional volume flow remains to be investigated by using numerical simulation, arguably the best approach for such investigations. The lattice Boltzmann method is used to study the CFS flow in a reconstructed geometry of the v3V. Simulation of CSF flow neglecting cilia in this geometry confirmed that the previous idea about pure confined flow does not reflect the reality observed in experiment. The experimentally recorded ciliary flow network along the wall was refined with the smoothed particle hydrodynamics and then adapted as boundary condition in simulation. We study the contribution of the ciliary network to overall CSF flow and identify site-specific delivery of CSF constituents with respect to the temporal changes.

Spatial distribution of calcium-gated chloride channels in olfactory cilia.

PubMed

French, Donald A; Badamdorj, Dorjsuren; Kleene, Steven J

2010-12-30

In vertebrate olfactory receptor neurons, sensory cilia transduce odor stimuli into changes in neuronal membrane potential. The voltage changes are primarily caused by the sequential openings of two types of channel: a cyclic-nucleotide-gated (CNG) cationic channel and a calcium-gated chloride channel. In frog, the cilia are 25 to 200 µm in length, so the spatial distributions of the channels may be an important determinant of odor sensitivity. To determine the spatial distribution of the chloride channels, we recorded from single cilia as calcium was allowed to diffuse down the length of the cilium and activate the channels. A computational model of this experiment allowed an estimate of the spatial distribution of the chloride channels. On average, the channels were concentrated in a narrow band centered at a distance of 29% of the ciliary length, measured from the base of the cilium. This matches the location of the CNG channels determined previously. This non-uniform distribution of transduction proteins is consistent with similar findings in other cilia. On average, the two types of olfactory transduction channel are concentrated in the same region of the cilium. This may contribute to the efficient detection of weak stimuli.

Loss of Dishevelleds disrupts planar polarity in ependymal motile cilia and results in hydrocephalus

PubMed Central

Ohata, Shinya; Nakatani, Jin; Herranz-Pérez, Vicente; Cheng, JrGang; Belinson, Haim; Inubushi, Toshiro; Snider, William D.; García-Verdugo, Jose Manuel; Wynshaw-Boris, Anthony; Álvarez-Buylla, Arturo

2014-01-01

SUMMARY Defects in ependymal (E) cells, which line the ventricle and generate cerebrospinal fluid flow through ciliary beating, can cause hydrocephalus. Dishevelled genes (Dvls) are essential for Wnt signaling and Dvl2 has been shown to localize to the rootlet of motile cilia. Using the hGFAP-Cre;Dvl1−/−;2flox/flox;3+/− mouse, we show that compound genetic ablation of Dvls causes hydrocephalus. In hGFAP-Cre;Dvl1−/−;2flox/flox;3+/− mutants, E cells differentiated normally, but the intracellular and intercellular rotational alignments of ependymal motile cilia were disrupted. As a consequence, the fluid flow generated by the hGFAP-Cre;Dvl1−/−;2flox/flox;3+/− E cells was significantly slower than that observed in control mice. Dvls were also required for the proper positioning of motile cilia on the apical surface. Tamoxifen-induced conditional removal of Dvls in adult mice also resulted in defects in intracellular rotational alignment and positioning of ependymal motile cilia. These results suggest that Dvls are continuously required for E cell planar polarity and may prevent hydrocephalus. PMID:25043421

The familial dysautonomia disease gene IKBKAP is required in the developing and adult mouse central nervous system

PubMed Central

Chaverra, Marta; George, Lynn; Thorne, Julian; Grindeland, Andrea; Ueki, Yumi; Eiger, Steven; Cusick, Cassie; Babcock, A. Michael; Carlson, George A.

2017-01-01

ABSTRACT Hereditary sensory and autonomic neuropathies (HSANs) are a genetically and clinically diverse group of disorders defined by peripheral nervous system (PNS) dysfunction. HSAN type III, known as familial dysautonomia (FD), results from a single base mutation in the gene IKBKAP that encodes a scaffolding unit (ELP1) for a multi-subunit complex known as Elongator. Since mutations in other Elongator subunits (ELP2 to ELP4) are associated with central nervous system (CNS) disorders, the goal of this study was to investigate a potential requirement for Ikbkap in the CNS of mice. The sensory and autonomic pathophysiology of FD is fatal, with the majority of patients dying by age 40. While signs and pathology of FD have been noted in the CNS, the clinical and research focus has been on the sensory and autonomic dysfunction, and no genetic model studies have investigated the requirement for Ikbkap in the CNS. Here, we report, using a novel mouse line in which Ikbkap is deleted solely in the nervous system, that not only is Ikbkap widely expressed in the embryonic and adult CNS, but its deletion perturbs both the development of cortical neurons and their survival in adulthood. Primary cilia in embryonic cortical apical progenitors and motile cilia in adult ependymal cells are reduced in number and disorganized. Furthermore, we report that, in the adult CNS, both autonomic and non-autonomic neuronal populations require Ikbkap for survival, including spinal motor and cortical neurons. In addition, the mice developed kyphoscoliosis, an FD hallmark, indicating its neuropathic etiology. Ultimately, these perturbations manifest in a developmental and progressive neurodegenerative condition that includes impairments in learning and memory. Collectively, these data reveal an essential function for Ikbkap that extends beyond the peripheral nervous system to CNS development and function. With the identification of discrete CNS cell types and structures that depend on Ikbkap, novel strategies to thwart the progressive demise of CNS neurons in FD can be developed. PMID:28167615

Simulations of centriole of polarized centrosome as a monopole antenna in immune and viral synapses.

PubMed

Dvorak, Josef; Melichar, Bohuslav; Filipova, Alzbeta; Grimova, Jana; Grimova, Nela; Rozsypalova, Aneta; Buka, David; Voboril, Rene; Zapletal, Radek; Buchler, Tomas; Richter, Igor; Buka, David

2018-01-01

The immune synapse (IS) is a temporary interface between an antigen-presenting cell and an effector lymphocyte. Viral synapse is a molecularly organized cellular junction that is structurally similar to the IS. Primary cilium is considered as a functional homologue of the IS due to the morphological and functional similarities in architecture between both micotubule structures. It has been hypothesized that endogenous electromagnetic field in the cell is generated by a unique cooperating system between mitochondria and microtubules. We are extending this prior hypothesis of the endogenous electromagnetic field in the cell postulating that polarized centriole in immune and viral synapse could serve as a monopole antenna. This is an addition to our hypothesis that primary cilium could serve as a monopole antenna. We simulated the distribution of electric field of centriole of polarized centrosome as a monopole antenna in immune and viral synapse. Very weak electromagnetic field of polarized centriole of CD8+ T lymphocyte in IS can contribute to the transport of cytolytic granules into the attacked (cancer) cell. Analogically, very weak electromagnetic field of polarized centriole in viral synapse of infected CD4 cells can aid the transport of viruses (human immunodeficiency virus) to non-infected CD4 cells. We hypothesized that healthy organisms need these monopole antennas. If, during the neoplastic transformation, healthy cells lose monopole antennas in form of primary cilia, the IS aims to replace them by monopole antennas of polarized centrioles in IS to restore homeostasis.

Drosophila Ana1 is required for centrosome assembly and centriole elongation.

PubMed

Saurya, Saroj; Roque, Hélio; Novak, Zsofia A; Wainman, Alan; Aydogan, Mustafa G; Volanakis, Adam; Sieber, Boris; Pinto, David Miguel Susano; Raff, Jordan W

2016-07-01

Centrioles organise centrosomes and cilia, and these organelles have an important role in many cell processes. In flies, the centriole protein Ana1 is required for the assembly of functional centrosomes and cilia. It has recently been shown that Cep135 (also known as Bld10) initially recruits Ana1 to newly formed centrioles, and that Ana1 then recruits Asl (known as Cep152 in mammals) to promote the conversion of these centrioles into centrosomes. Here, we show that ana1 mutants lack detectable centrosomes in vivo, that Ana1 is irreversibly incorporated into centrioles during their assembly and appears to play a more important role in maintaining Asl at centrioles than in initially recruiting Asl to centrioles. Unexpectedly, we also find that Ana1 promotes centriole elongation in a dose-dependent manner: centrioles are shorter when Ana1 dosage is reduced and are longer when Ana1 is overexpressed. This latter function of Ana1 appears to be distinct from its role in centrosome and cilium function, as a GFP-Ana1 fusion lacking the N-terminal 639 amino acids of the protein can support centrosome assembly and cilium function but cannot promote centriole over-elongation when overexpressed. © 2016. Published by The Company of Biologists Ltd.

Intraflagellar transport genes are essential for differentiation and survival of vertebrate sensory neurons.

PubMed

Tsujikawa, Motokazu; Malicki, Jarema

2004-06-10

Cilia play diverse roles in vertebrate and invertebrate sensory neurons. We show that a mutation of the zebrafish oval (ovl) locus affects a component of the ciliary transport (IFT) mechanism, the IFT88 polypeptide. In mutant retina, cilia are generated but not maintained, producing the absence of photoreceptor outer segments. A loss of cilia also occurs in auditory hair cells and olfactory sensory neurons. In all three sense organs, cilia defects are followed by degeneration of sensory cells. Similar phenotypes are induced by the absence of the IFT complex B polypeptides, ift52 and ift57, but not by the loss of complex A protein, ift140. The degeneration of mutant photoreceptor cells is caused, at least partially, by the ectopic accumulation of opsins. These studies reveal an essential role for IFT genes in vertebrate sensory neurons and implicate the molecular components of intraflagellar transport in degenerative disorders of these cells.

Nodal Cilia Dynamics and the Specification of the Left/Right Axis in Early Vertebrate Embryo Development

PubMed Central

Buceta, Javier; Ibañes, Marta; Rasskin-Gutman, Diego; Okada, Yasushi; Hirokawa, Nobutaka; Izpisúa-Belmonte, Juan Carlos

2005-01-01

Nodal cilia dynamics is a key factor for left/right axis determination in mouse embryos through the induction of a leftward fluid flow. So far it has not been clearly established how such dynamics is able to induce the asymmetric leftward flow within the node. Herein we propose that an asymmetric two-phase nonplanar beating cilia dynamics that involves the bending of the ciliar axoneme is responsible for the leftward fluid flow. We support our proposal with a host of hydrodynamic arguments, in silico experiments and in vivo video microscopy data in wild-type embryos and inv mutants. Our phenomenological modeling approach underscores how the asymmetry and speed of the flow depends on different relevant parameters. In addition, we discuss how the combination of internal and external mechanisms might cause the two-phase beating cilia dynamics. PMID:16040754

A Zebrafish Loss-of-Function Model for Human CFAP53 Mutations Reveals Its Specific Role in Laterality Organ Function.

PubMed

Noël, Emily S; Momenah, Tarek S; Al-Dagriri, Khalid; Al-Suwaid, Abdulrahman; Al-Shahrani, Safar; Jiang, Hui; Willekers, Sven; Oostveen, Yara Y; Chocron, Sonja; Postma, Alex V; Bhuiyan, Zahurul A; Bakkers, Jeroen

2016-02-01

Establishing correct left-right asymmetry during embryonic development is crucial for proper asymmetric positioning of the organs. Congenital heart defects, such as dextrocardia, transposition of the arteries, and inflow or outflow tract malformations, comprise some of the most common birth defects and may be attributed to incorrect establishment of body laterality. Here, we identify new patients with dextrocardia who have mutations in CFAP53, a coiled-coil domain containing protein. To elucidate the mechanism by which CFAP53 regulates embryonic asymmetry, we used genome editing to generate cfap53 zebrafish mutants. Zebrafish cfap53 mutants have specific defects in organ laterality and randomization of asymmetric gene expression. We show that cfap53 is required for cilia rotation specifically in Kupffer's vesicle, the zebrafish laterality organ, providing a mechanism by which patients with CFAP53 mutations develop dextrocardia and heterotaxy, and confirming previous evidence that left-right asymmetry in humans is regulated through cilia-driven fluid flow in a laterality organ. © 2015 WILEY PERIODICALS, INC.

Disease-Associated Mutations in CEP120 Destabilize the Protein and Impair Ciliogenesis.

PubMed

Joseph, Nimesh; Al-Jassar, Caezar; Johnson, Christopher M; Andreeva, Antonina; Barnabas, Deepak D; Freund, Stefan M V; Gergely, Fanni; van Breugel, Mark

2018-05-29

Ciliopathies are a group of genetic disorders caused by a failure to form functional cilia. Due to a lack of structural information, it is currently poorly understood how ciliopathic mutations affect protein functionality to give rise to the underlying disease. Using X-ray crystallography, we show that the ciliopathy-associated centriolar protein CEP120 contains three C2 domains. The point mutations V194A and A199P, which cause Joubert syndrome (JS) and Jeune asphyxiating thoracic dystrophy (JATD), respectively, both reduce the thermostability of the second C2 domain by targeting residues that point toward its hydrophobic core. Genome-engineered cells homozygous for these mutations have largely normal centriole numbers but show reduced CEP120 levels, compromised recruitment of distal centriole markers, and deficient cilia formation. Our results provide insight into the disease mechanism of two ciliopathic mutations in CEP120, identify putative binding partners of CEP120 C2B, and suggest a complex genotype-phenotype relation of the CEP120 ciliopathy alleles. Copyright © 2018 MRC Laboratory of Molecular Biology. Published by Elsevier Inc. All rights reserved.

BBSome function is required for both the morphogenesis and maintenance of the photoreceptor outer segment

PubMed Central

Hsu, Ying; Kim, Gunhee; Zhang, Qihong; Datta, Poppy; Seo, Seongjin

2017-01-01

Genetic mutations disrupting the structure and function of primary cilia cause various inherited retinal diseases in humans. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic ciliopathy characterized by retinal degeneration, obesity, postaxial polydactyly, intellectual disability, and genital and renal abnormalities. To gain insight into the mechanisms of retinal degeneration in BBS, we developed a congenital knockout mouse of Bbs8, as well as conditional mouse models in which function of the BBSome (a protein complex that mediates ciliary trafficking) can be temporally inactivated or restored. We demonstrate that BBS mutant mice have defects in retinal outer segment morphogenesis. We further demonstrate that removal of Bbs8 in adult mice affects photoreceptor function and disrupts the structural integrity of the outer segment. Notably, using a mouse model in which a gene trap inhibiting Bbs8 gene expression can be removed by an inducible FLP recombinase, we show that when BBS8 is restored in immature retinas with malformed outer segments, outer segment extension can resume normally and malformed outer segment discs are displaced distally by normal outer segment structures. Over time, the retinas of the rescued mice become morphologically and functionally normal, indicating that there is a window of plasticity when initial retinal outer segment morphogenesis defects can be ameliorated. PMID:29049287

Discerning the role of mechanosensors in regulating proximal tubule function

PubMed Central

Weisz, Ora A.

2015-01-01

All cells in the body experience external mechanical forces such as shear stress and stretch. These forces are sensed by specialized structures in the cell known as mechanosensors. Cells lining the proximal tubule (PT) of the kidney are continuously exposed to variations in flow rates of the glomerular ultrafiltrate, which manifest as changes in axial shear stress and radial stretch. Studies suggest that these cells respond acutely to variations in flow by modulating their ion transport and endocytic functions to maintain glomerulotubular balance. Conceptually, changes in the axial shear stress in the PT could be sensed by three known structures, namely, the microvilli, the glycocalyx, and primary cilia. The orthogonal component of the force produced by flow exhibits as radial stretch and can cause expansion of the tubule. Forces of stretch are transduced by integrins, by stretch-activated channels, and by cell-cell contacts. This review summarizes our current understanding of flow sensing in PT epithelia, discusses challenges in dissecting the role of individual flow sensors in the mechanosensitive responses, and identifies potential areas of opportunity for new study. PMID:26662200

Kupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut.

PubMed

Essner, Jeffrey J; Amack, Jeffrey D; Nyholm, Molly K; Harris, Erin B; Yost, H Joseph

2005-03-01

Monocilia have been proposed to establish the left-right (LR) body axis in vertebrate embryos by creating a directional fluid flow that triggers asymmetric gene expression. In zebrafish, dorsal forerunner cells (DFCs) express a conserved ciliary dynein gene (left-right dynein-related1, lrdr1) and form a ciliated epithelium inside a fluid-filled organ called Kupffer's vesicle (KV). Here, videomicroscopy demonstrates that cilia inside KV are motile and create a directional fluid flow just prior to the onset of asymmetric gene expression in lateral cells. Laser ablation of DFCs and surgical disruption of KV provide direct evidence that ciliated KV cells are required during early somitogenesis for subsequent LR patterning in the brain, heart and gut. Antisense morpholinos against lrdr1 disrupt KV fluid flow and perturb LR development. Furthermore, lrdr1 morpholinos targeted to DFC/KV cells demonstrate that Lrdr1 functions in these ciliated cells to control LR patterning. This provides the first direct evidence, in any vertebrate, that impairing cilia function in derivatives of the dorsal organizer, and not in other cells that express ciliogenic genes, alters LR development. Finally, genetic analysis reveals novel roles for the T-box transcription factor no tail and the Nodal signaling pathway as upstream regulators of lrdr1 expression and KV morphogenesis. We propose that KV is a transient embryonic 'organ of asymmetry' that directs LR development by establishing a directional fluid flow. These results suggest that cilia are an essential component of a conserved mechanism that controls the transition from bilateral symmetry to LR asymmetry in vertebrates.

Morphometric and ultrastructural comparison of the olfactory system in elasmobranchs: the significance of structure-function relationships based on phylogeny and ecology.

PubMed

Schluessel, Vera; Bennett, Michael B; Bleckmann, Horst; Blomberg, Simon; Collin, Shaun P

2008-11-01

This study investigated the relationship between olfactory morphology, habitat occupancy, and lifestyle in 21 elasmobranch species in a phylogenetic context. Four measures of olfactory capability, that is, the number of olfactory lamellae, the surface area of the olfactory epithelium, the mass of the olfactory bulb, and the mass of the olfactory rosette were compared between individual species and groups, comprised of species with similar habitat and/or lifestyle. Statistical analyses using generalized least squares phylogenetic regression revealed that bentho-pelagic sharks and rays possess significantly more olfactory lamellae and larger sensory epithelial surface areas than benthic species. There was no significant correlation between either olfactory bulb or rosette mass and habitat type. There was also no significant difference between the number of lamellae or the size of the sensory surface area in groups comprised of species with similar diets, that is, groups preying predominantly on crustaceans, cephalopods, echinoderms, polychaetes, molluscs, or teleosts. However, some groups had significantly larger olfactory bulb or rosette masses than others. There was little evidence to support a correlation between phylogeny and morphology, indicating that differences in olfactory capabilities are the result of functional rather than phylogenetic adaptations. All olfactory epithelia exhibited microvilli and cilia, with microvilli in both nonsensory and sensory areas, and cilia only in sensory areas. Cilia over the sensory epithelia originated from supporting cells. In contrast to teleosts, which possess ciliated and microvillous olfactory receptor types, no ciliated olfactory receptor cells were observed. This is the first comprehensive study comparing olfactory morphology to several aspects of elasmobranch ecology in a phylogenetic context.

Distinct functions for IFT140 and IFT20 in opsin transport.

PubMed Central

Crouse, Jacquelin A.; Lopes, Vanda S.; SanAgustin, Jovenal T.; Keady, Brian T.; Williams, David S.; Pazour, Gregory J.

2014-01-01

In the vertebrate retina, light is detected by the outer segments of photoreceptor rods and cones, which are highly modified cilia. Like other cilia, outer segments have no protein synthetic capacity and depend on proteins made in the cell body for their formation and maintenance. The mechanism of transport into the outer segment is not fully understood but intraflagellar transport (IFT) is thought to be a major mechanism for moving protein from the cell body into the cilium. In the case of photoreceptor cells, the high density of receptors and the disk turnover that occurs daily necessitates much higher rates of transport than would be required in other cilia. In this work, we show that the IFT complex A protein IFT140 is required for development and maintenance of outer segments. In earlier work we found that acute deletion of Ift20 caused opsin to accumulate at the Golgi complex. In this work we find that acute deletion of Ift140 does not cause opsin to accumulate at the Golgi complex but rather it accumulates in the plasma membrane of the inner segments. This work is strong support of a model of opsin transport where IFT20 is involved in the movement from the Golgi complex to the base of the cilium. Then, once at the base, the opsin is carried through the connecting cilium by an IFT complex that includes IFT140. PMID:24619649

Does vitamin E prevent tubal damage caused by smoking? A light microscopy and animal study.

PubMed

Duran, Muzeyyen; Ustunyurt, Emin; Kosus, Aydin; Kosus, Nermin; Turhan, Nilgun; Hızlı, Deniz; Sarac, Gulce Naz; Erdogan, Deniz

2014-04-01

To assess the histomorphological effects of smoking on the cilia of fallopian tubes in mice and the effect of vitamin E on the negative effects of smoke. Eighteen 12-14 week-old Swiss albino type female mice were randomly divided into three groups, each consisting of six mice: Group A: control group; Group B: mice exposed to cigarette smoke; Group C: mice exposed to cigarette smoke together with vitamin E. Groups B and C were exposed to cigarette smoke for 10 weeks. After 10 weeks, tubal excision was performed in all animals. Histopathologic examination of excised tubal tissue was conducted under light microscopy. The number of cilia was significantly lower in Group B. Although not statistically significant, the median number of cilia in Group C was measured to be higher than in Group B but lower than in Group A. Based on the results, it can be concluded that smoking decreases tubal cilia numbers. Supplementation by vitamin E may treat or at least help to slow down the decrease in number of cilia caused by smoking; therefore it could be used therapeutically in the treatment of smoking-related tubal damage. Crown Copyright © 2014. Published by Elsevier Ireland Ltd. All rights reserved.

Levonorgestrel decreases cilia beat frequency of human fallopian tubes and rat oviducts without changing morphological structure.

PubMed

Zhao, Weihong; Zhu, Qian; Yan, Mingxing; Li, Cheng; Yuan, Jiangjing; Qin, Guojuan; Zhang, Jian

2015-02-01

Levonorgestrel, a derivative of progesterone, effectively protects women against unwanted pregnancy as an emergency contraceptive. Previous studies have not been successful in determining the mechanism by which levonorgestrel acts. In the present study we analysed cilia beat action and cilia morphology following levonorgestrel exposure in vitro and in vivo using both light and electron microscopy. There was a significant decrease in the ciliary beat frequency (CBF) of human fallopian tubes between mucosal explants bathed in 5 μmol/L levonorgestrel and those bathed in medium alone (P < 0.05). There was a tendency for CBF to decrease more in the ampulla than in isthmus, but there were no differences between the proliferative and secretory phases. In rat oviducts, levonorgestrel produced a similar reduction in CBF (~ 10%) compared with the saline control group (P < 0.05). Histological and ultrastructural analysis demonstrated no changes in the percentage of ciliated cells or in the classic '9 + 2' structure of cilia following levonorgestrel treatment in either system. Thus, levonorgestrel reduces CBF without damaging cilia morphology. Decreases in CBF may indicate a pathological role for levonorgestrel in the transportation of the ovum and zygote in the fallopian tube. © 2014 Wiley Publishing Asia Pty Ltd.

An Outer Arm Dynein Conformational Switch Is Required for Metachronal Synchrony of Motile Cilia in Planaria

PubMed Central

Rompolas, Panteleimon; Patel-King, Ramila S.

2010-01-01

Motile cilia mediate the flow of mucus and other fluids across the surface of specialized epithelia in metazoans. Efficient clearance of peri-ciliary fluids depends on the precise coordination of ciliary beating to produce metachronal waves. The role of individual dynein motors and the mechanical feedback mechanisms required for this process are not well understood. Here we used the ciliated epithelium of the planarian Schmidtea mediterranea to dissect the role of outer arm dynein motors in the metachronal synchrony of motile cilia. We demonstrate that animals that completely lack outer dynein arms display a significant decline in beat frequency and an inability of cilia to coordinate their oscillations and form metachronal waves. Furthermore, lack of a key mechanosensitive regulatory component (LC1) yields a similar phenotype even though outer arms still assemble in the axoneme. The lack of metachrony was not due simply to a decrease in ciliary beat frequency, as reducing this parameter by altering medium viscosity did not affect ciliary coordination. In addition, we did not observe a significant temporal variability in the beat cycle of impaired cilia. We propose that this conformational switch provides a mechanical feedback system within outer arm dynein that is necessary to entrain metachronal synchrony. PMID:20844081

Paramecium swimming and ciliary beating patterns: a study on four RNA interference mutations.

PubMed

Funfak, Anette; Fisch, Cathy; Abdel Motaal, Hatem T; Diener, Julien; Combettes, Laurent; Baroud, Charles N; Dupuis-Williams, Pascale

2015-01-01

Paramecium cells swim and feed by beating their thousands of cilia in coordinated patterns. The organization of these patterns and its relationship with cell motility has been the subject of a large body of work, particularly as a model for ciliary beating in human organs where similar organization is seen. However the rapid motion of the cells makes quantitative measurements very challenging. Here we provide detailed measurements of the swimming of Paramecium cells from high-speed video at high magnification, as they move in microfluidic channels. An image analysis protocol allows us to decouple the cell movement from the motion of the cilia, thus allowing us to measure the ciliary beat frequency (CBF) and the spatio-temporal organization into metachronal waves along the cell periphery. Two distinct values of the CBF appear at different regions of the cell: most of the cilia beat in the range of 15 to 45 Hz, while the cilia in the peristomal region beat at almost double the frequency. The body and peristomal CBF display a nearly linear relation with the swimming velocity. Moreover the measurements do not display a measurable correlation between the swimming velocity and the metachronal wave velocity on the cell periphery. These measurements are repeated for four RNAi silenced mutants, where proteins specific to the cilia or to their connection to the cell base are depleted. We find that the mutants whose ciliary structure is affected display similar swimming to the control cells albeit with a reduced efficiency, while the mutations that affect the cilia's anchoring to the cell lead to strongly reduced ability to swim. This reduction in motility can be related to a loss of coordination between the ciliary beating in different parts of the cell.

Synthesis and Turnover of Embryonic Sea Urchin Ciliary Proteins during Selective Inhibition of Tubulin Synthesis and Assembly

PubMed Central

Stephens, Raymond E.

1997-01-01

When ciliogenesis first occurs in sea urchin embryos, the major building block proteins, tubulin and dynein, exist in substantial pools, but most 9+2 architectural proteins must be synthesized de novo. Pulse-chase labeling with [3H]leucine demonstrates that these proteins are coordinately up-regulated in response to deciliation so that regeneration ensues and the tubulin and dynein pools are replenished. Protein labeling and incorporation into already-assembled cilia is high, indicating constitutive ciliary gene expression and steady-state turnover. To determine whether either the synthesis of tubulin or the size of its available pool is coupled to the synthesis or turnover of the other 9+2 proteins in some feedback manner, fully-ciliated mid- or late-gastrula stage Strongylocentrotus droebachiensis embryos were pulse labeled in the presence of colchicine or taxol at concentrations that block ciliary growth. As a consequence of tubulin autoregulation mediated by increased free tubulin, no labeling of ciliary tubulin occurred in colchicine-treated embryos. However, most other proteins were labeled and incorporated into steady-state cilia at near-control levels in the presence of colchicine or taxol. With taxol, tubulin was labeled as well. An axoneme-associated 78 kDa cognate of the molecular chaperone HSP70 correlated with length during regeneration; neither colchicine nor taxol influenced the association of this protein in steady-state cilia. These data indicate that 1) ciliary protein synthesis and turnover is independent of tubulin synthesis or tubulin pool size; 2) steady-state incorporation of labeled proteins cannot be due to formation or elongation of cilia; 3) substantial tubulin exchange takes place in fully-motile cilia; and 4) chaperone presence and association in steady-state cilia is independent of background ciliogenesis, tubulin synthesis, and tubulin assembly state. PMID:9362062

The effects of the stem cell on ciliary regeneration of injured rabbit sinonasal epithelium.

PubMed

Kavuzlu, Ali; Tatar, Emel Çadallı; Karagöz, Tuğba; Pınarlı, Ferda Alpaslan; Tatar, İlkan; Bayır, Ömer; Korkmaz, Mehmet Hakan

2017-08-01

Defects in mucosal healing after sinonasal surgery cause infection, scar formation causing obstruction, relapse of the disease within a shorter period and revision surgery. The present study aimed to create a functional ciliated epithelium using a stem cell and stem cell sheet of adipose tissue origin and to show such regeneration ultra-structurally on experimentally injured rabbit nasal epithelium. This was an experimental animal study and basic research. A total of 18 white New Zealand rabbits were divided into three groups. The medial wall of the maxillary sinus of the subjects was peeled off bilaterally. No additional procedure was applied to the subjects in Group 1. In Group 2, adipose tissue-derived mesenchymal stem cell was implanted on the wound edges of the subjects. In Group 3, a stem cell sheet of three layers was laid onto the defect area. All subjects were killed after 3 weeks. The presence of the stem cell stained with bromo-deoxyuridine was assessed with a light microscope, whereas cilia density, ciliated orientation and cilia structure were evaluated with a scanning electron microscope. Ciliary densities in Group 2 and Group 3 were statistically superior compared to the control group (p < 0.001, p = 0.007). Cilia morphology in Group 2 and Group 3 was also better than the control group (p < 0.01, p = 0.048). Ciliary orientation in Group 2 was scored highest (p < 0.01). The ratio of BrDu-stained cells was observed to be 27% in Group 3 and 8% in Group 2. Sub-epithelial recovery was observed to be better in Group 3. Adipose tissue-derived mesenchymal stem cell increased the healing of the injured maxillary sinus mucosa of the rabbits in terms of cilia presence, density and morphology regardless of the implementation technique. Level of evidence NA.

Interfacial distribution of mucus under forced expiration in a double bifurcation model

NASA Astrophysics Data System (ADS)

Rajendran, Rahul; Banerjee, Arindam

2017-11-01

Mucus is removed from the lung airways by the rhythmic beating of cilia and the mucus interaction with the turbulent core airflow generated during a cough or forced expiration. The quantity and quality of mucus are adversely altered, impairing mucociliary clearance under chronic pulmonary conditions. Existing studies on airflow induced mucus clearance have established a functional relationship between the airflow rate, mucus properties, flow bias, breathing frequency and clearance; however, the impact of airway branching, gravity, and characterization of primary and secondary flows have not been studied. The focus of the current investigation is the detailed understanding of air-mucus two-phase flow mechanism under steady expiratory airflow in a double bifurcation model. The effect of different airflow rates and mucus viscosities on the flow morphology, mucus layer thickness, mucus clearance and pressure drop across the model will be discussed. The impact of in-plane and out-of-plane configurations of the bifurcation model on the primary and secondary flow structures as well as the mucus distribution will be addressed. In addition, a detailed comparison of the flow structures in the mucus-lined airways, and its corresponding dry wall (no mucus lining) case will be presented.

Automated software for analysis of ciliary beat frequency and metachronal wave orientation in primary ciliary dyskinesia.

PubMed

Mantovani, Giulia; Pifferi, Massimo; Vozzi, Giovanni

2010-06-01

Patients with primary ciliary dyskinesia (PCD) have structural and/or functional alterations of cilia that imply deficits in mucociliary clearance and different respiratory pathologies. A useful indicator for the difficult diagnosis is the ciliary beat frequency (CBF) that is significantly lower in pathological cases than in physiological ones. The CBF computation is not rapid, therefore, the aim of this study is to propose an automated method to evaluate it directly from videos of ciliated cells. The cells are taken from inferior nasal turbinates and videos of ciliary movements are registered and eventually processed by the developed software. The software consists in the extraction of features from videos (written with C++ language) and the computation of the frequency (written with Matlab language). This system was tested both on the samples of nasal cavity and software models, and the results were really promising because in a few seconds, it can compute a reliable frequency if compared with that measured with visual methods. It is to be noticed that the reliability of the computation increases with the quality of acquisition system and especially with the sampling frequency. It is concluded that the developed software could be a useful mean for PCD diagnosis.

Bardet-Biedl Syndrome

PubMed Central

Suspitsin, Evgeny N.; Imyanitov, Evgeny N.

2016-01-01

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive genetic disorder. It is characterized by heterogeneous clinical manifestations including primary features of the disease (rod-cone dystrophy, polydactyly, obesity, genital abnormalities, renal defects, and learning difficulties) and secondary BBS characteristics (developmental delay, speech deficit, brachydactyly or syndactyly, dental defects, ataxia or poor coordination, olfactory deficit, diabetes mellitus, congenital heart disease, etc.); most of these symptoms may not be present at birth but appear and progressively worsen during the first and second decades of life. At least 20 BBS genes have already been identified, and all of them are involved in primary cilia functioning. Genetic diagnosis of BBS is complicated due to lack of gene-specific disease symptoms; however, it is gradually becoming more accessible with the invention of multigene sequencing technologies. Clinical management of BBS is largely limited to a symptomatic treatment. Mouse experiments demonstrate that the most debilitating complication of BBS, blindness, can be rescued by topical gene therapy. There is a published case report describing the delay of BBS symptoms by nutritional compensation of the disease-related biochemical deficiencies. Progress in DNA testing technologies is likely to rapidly resolve all limitations in BBS diagnosis; however, much slower improvement is expected with regard to BBS treatment. PMID:27385962

Expression of Nek1 during kidney development and cyst formation in multiple nephron segments in the Nek1-deficient kat2J mouse model of polycystic kidney disease.

PubMed

Chen, Yumay; Chiang, Huai-Chin; Litchfield, Patricia; Pena, Michelle; Juang, Charity; Riley, Daniel J

2014-07-17

Neks, mammalian orthologs of the fungal protein kinase never-in-mitosis A, have been implicated in the pathogenesis of polycystic kidney disease. Among them, Nek1 is the primary protein inactivated in kat2J mouse models of PKD. We report the expression pattern of Nek1 and characterize the renal cysts that develop in kat2J mice. Nek1 is detectable in all murine tissues but its expression in wild type and kat2J heterozygous kidneys decrease as the kidneys mature, especially in tubular epithelial cells. In the embryonic kidney, Nek1 expression is most prominent in cells that will become podocytes and proximal tubules. Kidney development in kat2J homozygous mice is aberrant early, before the appearance of gross cysts: developing cortical zones are thin, populated by immature glomeruli, and characterized by excessive apoptosis of several cell types. Cysts in kat2J homozygous mice form postnatally in Bowman's space as well as different tubular subtypes. Late in life, kat2J heterozygous mice form renal cysts and the cells lining these cysts lack staining for Nek1. The primary cilia of cells lining cysts in kat2J homozygous mice are morphologically diverse: in some cells they are unusually long and in others there are multiple cilia of varying lengths. Our studies indicate that Nek1 deficiency leads to disordered kidney maturation, and cysts throughout the nephron.

Cftr controls lumen expansion and function of Kupffer’s vesicle in zebrafish

PubMed Central

Navis, Adam; Marjoram, Lindsay; Bagnat, Michel

2013-01-01

Regulated fluid secretion is crucial for the function of most organs. In vertebrates, the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) is a master regulator of fluid secretion. Although the biophysical properties of CFTR have been well characterized in vitro, little is known about its in vivo role during development. Here, we investigated the function of Cftr during zebrafish development by generating several cftr mutant alleles using TAL effector nucleases. We found that loss of cftr function leads to organ laterality defects. In zebrafish, left-right (LR) asymmetry requires cilia-driven fluid flow within the lumen of Kupffer’s vesicle (KV). Using live imaging we found that KV morphogenesis is disrupted in cftr mutants. Loss of Cftr-mediated fluid secretion impairs KV lumen expansion leading to defects in organ laterality. Using bacterial artificial chromosome recombineering, we generated transgenic fish expressing functional Cftr fusion proteins with fluorescent tags under the control of the cftr promoter. The transgenes completely rescued the cftr mutant phenotype. Live imaging of these transgenic lines showed that Cftr is localized to the apical membrane of the epithelial cells in KV during lumen formation. Pharmacological stimulation of Cftr-dependent fluid secretion led to an expansion of the KV lumen. Conversely, inhibition of ion gradient formation impaired KV lumen inflation. Interestingly, cilia formation and motility in KV were not affected, suggesting that fluid secretion and flow are independently controlled in KV. These findings uncover a new role for cftr in KV morphogenesis and function during zebrafish development. PMID:23487313

MKS5 and CEP290 Dependent Assembly Pathway of the Ciliary Transition Zone

PubMed Central

Li, Chunmei; Kennedy, Julie; Garcia-Gonzalo, Francesc R.; Romani, Marta; De Mori, Roberta; Bruel, Ange-Line; Gaillard, Dominique; Doray, Bérénice; Lopez, Estelle; Rivière, Jean-Baptiste; Faivre, Laurence; Thauvin-Robinet, Christel; Reiter, Jeremy F.; Blacque, Oliver E.; Valente, Enza Maria; Leroux, Michel R.

2016-01-01

Cilia have a unique diffusion barrier (“gate”) within their proximal region, termed transition zone (TZ), that compartmentalises signalling proteins within the organelle. The TZ is known to harbour two functional modules/complexes (Meckel syndrome [MKS] and Nephronophthisis [NPHP]) defined by genetic interaction, interdependent protein localisation (hierarchy), and proteomic studies. However, the composition and molecular organisation of these modules and their links to human ciliary disease are not completely understood. Here, we reveal Caenorhabditis elegans CEP-290 (mammalian Cep290/Mks4/Nphp6 orthologue) as a central assembly factor that is specific for established MKS module components and depends on the coiled coil region of MKS-5 (Rpgrip1L/Rpgrip1) for TZ localisation. Consistent with a critical role in ciliary gate function, CEP-290 prevents inappropriate entry of membrane-associated proteins into cilia and keeps ARL-13 (Arl13b) from leaking out of cilia via the TZ. We identify a novel MKS module component, TMEM-218 (Tmem218), that requires CEP-290 and other MKS module components for TZ localisation and functions together with the NPHP module to facilitate ciliogenesis. We show that TZ localisation of TMEM-138 (Tmem138) and CDKL-1 (Cdkl1/Cdkl2/Cdkl3/Cdlk4 related), not previously linked to a specific TZ module, similarly depends on CEP-290; surprisingly, neither TMEM-138 or CDKL-1 exhibit interdependent localisation or genetic interactions with core MKS or NPHP module components, suggesting they are part of a distinct, CEP-290-associated module. Lastly, we show that families presenting with Oral-Facial-Digital syndrome type 6 (OFD6) have likely pathogenic mutations in CEP-290-dependent TZ proteins, namely Tmem17, Tmem138, and Tmem231. Notably, patient fibroblasts harbouring mutated Tmem17, a protein not yet ciliopathy-associated, display ciliogenesis defects. Together, our findings expand the repertoire of MKS module-associated proteins—including the previously uncharacterised mammalian Tmem80—and suggest an MKS-5 and CEP-290-dependent assembly pathway for building a functional TZ. PMID:26982032

Physical limits of flow sensing in the left-right organizer

PubMed Central

Ferreira, Rita R; Vilfan, Andrej; Jülicher, Frank; Supatto, Willy; Vermot, Julien

2017-01-01

Fluid flows generated by motile cilia are guiding the establishment of the left-right asymmetry of the body in the vertebrate left-right organizer. Competing hypotheses have been proposed: the direction of flow is sensed either through mechanosensation, or via the detection of chemical signals transported in the flow. We investigated the physical limits of flow detection to clarify which mechanisms could be reliably used for symmetry breaking. We integrated parameters describing cilia distribution and orientation obtained in vivo in zebrafish into a multiscale physical study of flow generation and detection. Our results show that the number of immotile cilia is too small to ensure robust left and right determination by mechanosensing, given the large spatial variability of the flow. However, motile cilia could sense their own motion by a yet unknown mechanism. Finally, transport of chemical signals by the flow can provide a simple and reliable mechanism of asymmetry establishment. DOI: http://dx.doi.org/10.7554/eLife.25078.001 PMID:28613157

Cilia-like structures anchor the amphioxus notochord to its sheath.

PubMed

Bočina, Ivana; Ljubešić, Nikola; Saraga-Babić, Mirna

2011-01-01

Body stiffness is important during undulatory locomotion in fish. In amphioxus, the myosepta play an important role in transmission of muscular forces to the notochord. In order to define the specific supporting role of the notochord in amphioxus during locomotion, the ultrastructure of 10 adult amphioxus specimens was analyzed using transmission electron microscopy. Numerous cilia-like structures were found on the surface of each notochordal cell at the sites of their attachment to the notochordal sheath. Ultrastructurally, these structures consisted of the characteristic arrangement of peripheral and central microtubular doublets and were anchored to the inner layer of the notochordal sheath. Immunohistochemically, a positive reaction to applied dynein and β-tubulin antibodies characterized the area of the cilia-like structures. We propose that reduced back-and-forth movements of the cilia-like structures might contribute to the flow of the fluid content inside the notochord, thus modulating the stiffness of the amphioxus body during its undulatory locomotion. Copyright © 2009 Elsevier GmbH. All rights reserved.

Neural network detected in a presumed vestigial trait: ultrastructure of the salmonid adipose fin

PubMed Central

Buckland-Nicks, J. A.; Gillis, M.; Reimchen, T. E.

2012-01-01

A wide variety of rudimentary and apparently non-functional traits have persisted over extended evolutionary time. Recent evidence has shown that some of these traits may be maintained as a result of developmental constraints or neutral energetic cost, but for others their true function was not recognized. The adipose fin is small, fleshy, non-rayed and located between the dorsal and caudal fins on eight orders of basal teleosts and has traditionally been regarded as vestigial without clear function. We describe here the ultrastructure of the adipose fin and for the first time, to our knowledge, present evidence of extensive nervous tissue, as well as an unusual subdermal complex of interconnected astrocyte-like cells equipped with primary cilia. The fin contains neither adipose tissue nor fin rays. Many fusiform actinotrichia, comprising dense striated macrofibrils, support the free edge and connect with collagen cables that link the two sides. These results are consistent with a recent hypothesis that the adipose fin may act as a precaudal flow sensor, where its removal can be detrimental to swimming efficiency in turbulent water. Our findings provide insight to the broader themes of function versus constraints in evolutionary biology and may have significance for fisheries science, as the adipose fin is routinely removed from millions of salmonids each year. PMID:21733904

Primary cilium - antenna-like structure on the surface of most mammalian cell types

NASA Astrophysics Data System (ADS)

Dvorak, J.; Sitorova, V.; Hadzi Nikolov, D.; Mokry, J.; Richter, I.; Kasaova, L.; Filip, S.; Ryska, A.; Petera, J.

2011-12-01

The primary cilium is a sensory solitary non-motile microtubule-based organelle protruding in the quiescent phase of the cell cycle from the surface of the majority of human cells, including embryonic cells, stem cells and stromal cells of malignant tumors. The presence of a primary cilium on the surface of a cell is transient, limited to the quiescent G1(G0) phase and the beginning of the S phase of the cell cycle. The primary cilium is formed from the mother centriole. Primary cilia are key coordinators of signaling pathways during development and tissue homeostasis and, when deffective, they are a major cause of human diseases and developmental disorders, now commonly referred to as ciliopathies. Most cancer cells do not possess a primary cilium. The loss of the primary cilium is a regular feature of neoplastic transformation in the majority of solid tumors. The primary cilium could serve as a tumor suppressor organelle. The aim of this paper was to provide a review of the current knowledge of the primary cilium.

Use of a Novel Cell Adhesion Method and Digital Measurement to Show Stimulus-dependent Variation in Somatic and Oral Ciliary Beat Frequency in Paramecium

PubMed Central

Bell, Wade E.; Hallworth, Richard; Wyatt, Todd A.; Sisson, Joseph H.

2015-01-01

When Paramecium encounters positive stimuli, the membrane hyperpolarizes and ciliary beat frequency increases. We adapted an established immobilization protocol using a biological adhesive and a novel digital analysis system to quantify beat frequency in immobilized Paramecium. Cells showed low mortality and demonstrated beat frequencies consistent with previous studies. Chemoattractant molecules, reduction in external potassium, and posterior stimulation all increased somatic beat frequency. In all cases, the oral groove cilia maintained a higher beat frequency than mid-body cilia, but only oral cilia from cells stimulated with chemoattactants showed an increase from basal levels. PMID:25066640

Targeting Midbodies in Ovarian Cancer Stem Cells as a Therapeutic Strategy

DTIC Science & Technology

2013-10-01

functional spindle assembly. Nat. Cell Biol. 13, 1406–1414. Ori-McKenney, K.M., Jan, L.Y., and Jan, Y.-N. (2012). Golgi outposts shape dendrite morphology...1899. Yadav, S., and Linstedt, A.D. (2011). Golgi positioning. Cold Spring Harb. Perspect . Biol. 3, 3. Zhang, H., Squirrell, J.M., and White, J.G. (2008...implications for recy- cling endosome function . This new liaison has additional impli- cations for a variety of biological processes including cilia

Centrioles want to move out and make cilia.

PubMed

Pearson, Chad G; Culver, Brady P; Winey, Mark

2007-09-01

Cilia formation in mammalian cells requires basal bodies that are either derived from centrioles that transition from their cytoplasmic role in centrosome organization or that form en masse in multiciliated cells. Several recent studies have begun to uncover the links between centriole duplication and their transformation to basal bodies.

Formation of the transition zone by Mks5/Rpgrip1L establishes a ciliary zone of exclusion (CIZE) that compartmentalises ciliary signalling proteins and controls PIP2 ciliary abundance

PubMed Central

Jensen, Victor L; Li, Chunmei; Bowie, Rachel V; Clarke, Lara; Mohan, Swetha; Blacque, Oliver E; Leroux, Michel R

2015-01-01

Cilia are thought to harbour a membrane diffusion barrier within their transition zone (TZ) that compartmentalises signalling proteins. How this “ciliary gate” assembles and functions remains largely unknown. Contrary to current models, we present evidence that Caenorhabditis elegans MKS-5 (orthologue of mammalian Mks5/Rpgrip1L/Nphp8 and Rpgrip1) may not be a simple structural scaffold for anchoring > 10 different proteins at the TZ, but instead, functions as an assembly factor. This activity is needed to form TZ ultrastructure, which comprises Y-shaped axoneme-to-membrane connectors. Coiled-coil and C2 domains within MKS-5 enable TZ localisation and functional interactions with two TZ modules, consisting of Meckel syndrome (MKS) and nephronophthisis (NPHP) proteins. Discrete roles for these modules at basal body-associated transition fibres and TZ explain their redundant functions in making essential membrane connections and thus sealing the ciliary compartment. Furthermore, MKS-5 establishes a ciliary zone of exclusion (CIZE) at the TZ that confines signalling proteins, including GPCRs and NPHP-2/inversin, to distal ciliary subdomains. The TZ/CIZE, potentially acting as a lipid gate, limits the abundance of the phosphoinositide PIP2 within cilia and is required for cell signalling. Together, our findings suggest a new model for Mks5/Rpgrip1L in TZ assembly and function that is essential for establishing the ciliary signalling compartment. PMID:26392567

Truncation of POC1A associated with short stature and extreme insulin resistance.

PubMed

Chen, Jian-Hua; Segni, Maria; Payne, Felicity; Huang-Doran, Isabel; Sleigh, Alison; Adams, Claire; Savage, David B; O'Rahilly, Stephen; Semple, Robert K; Barroso, Inês

2015-10-01

We describe a female proband with primordial dwarfism, skeletal dysplasia, facial dysmorphism, extreme dyslipidaemic insulin resistance and fatty liver associated with a novel homozygous frameshift mutation in POC1A, predicted to affect two of the three protein products of the gene. POC1A encodes a protein associated with centrioles throughout the cell cycle and implicated in both mitotic spindle and primary ciliary function. Three homozygous mutations affecting all isoforms of POC1A have recently been implicated in a similar syndrome of primordial dwarfism, although no detailed metabolic phenotypes were described. Primary cells from the proband we describe exhibited increased centrosome amplification and multipolar spindle formation during mitosis, but showed normal DNA content, arguing against mitotic skipping, cleavage failure or cell fusion. Despite evidence of increased DNA damage in cells with supernumerary centrosomes, no aneuploidy was detected. Extensive centrosome clustering both at mitotic spindles and in primary cilia mitigated the consequences of centrosome amplification, and primary ciliary formation was normal. Although further metabolic studies of patients with POC1A mutations are warranted, we suggest that POC1A may be added to ALMS1 and PCNT as examples of centrosomal or pericentriolar proteins whose dysfunction leads to extreme dyslipidaemic insulin resistance. Further investigation of links between these molecular defects and adipose tissue dysfunction is likely to yield insights into mechanisms of adipose tissue maintenance and regeneration that are critical to metabolic health. © 2015 Society for Endocrinology.

Small organelle, big responsibility: the role of centrosomes in development and disease

PubMed Central

Chavali, Pavithra L.; Pütz, Monika; Gergely, Fanni

2014-01-01

The centrosome, a key microtubule organizing centre, is composed of centrioles, embedded in a protein-rich matrix. Centrosomes control the internal spatial organization of somatic cells, and as such contribute to cell division, cell polarity and migration. Upon exiting the cell cycle, most cell types in the human body convert their centrioles into basal bodies, which drive the assembly of primary cilia, involved in sensing and signal transduction at the cell surface. Centrosomal genes are targeted by mutations in numerous human developmental disorders, ranging from diseases exclusively affecting brain development, through global growth failure syndromes to diverse pathologies associated with ciliary malfunction. Despite our much-improved understanding of centrosome function in cellular processes, we know remarkably little of its role in the organismal context, especially in mammals. In this review, we examine how centrosome dysfunction impacts on complex physiological processes and speculate on the challenges we face when applying knowledge generated from in vitro and in vivo model systems to human development. PMID:25047622

KIF7 mutations cause fetal hydrolethalus and acrocallosal syndromes

PubMed Central

Putoux, Audrey; Thomas, Sophie; Coene, Karlien L M; Davis, Erica E; Alanay, Yasemin; Ogur, Gönül; Uz, Elif; Buzas, Daniela; Gomes, Céline; Patrier, Sophie; Bennett, Christopher L; Elkhartoufi, Nadia; Frison, Marie-Hélène Saint; Rigonnot, Luc; Joyé, Nicole; Pruvost, Solenn; Utine, Gulen Eda; Boduroglu, Koray; Nitschke, Patrick; Fertitta, Laura; Thauvin-Robinet, Christel; Munnich, Arnold; Cormier-Daire, Valérie; Hennekam, Raoul; Colin, Estelle; Akarsu, Nurten Ayse; Bole-Feysot, Christine; Cagnard, Nicolas; Schmitt, Alain; Goudin, Nicolas; Lyonnet, Stanislas; Encha-Razavi, Férechté; Siffroi, Jean-Pierre; Winey, Mark; Katsanis, Nicholas; Gonzales, Marie; Vekemans, Michel; Beales, Philip L; Attié-Bitach, Tania

2012-01-01

KIF7, the human ortholog of Drosophila Costal2, is a key component of the Hedgehog signaling pathway. Here we report mutations in KIF7 in individuals with hydrolethalus and acrocallosal syndromes, two multiple malformation disorders with overlapping features that include polydactyly, brain abnormalities and cleft palate. Consistent with a role of KIF7 in Hedgehog signaling, we show deregulation of most GLI transcription factor targets and impaired GLI3 processing in tissues from individuals with KIF7 mutations. KIF7 is also a likely contributor of alleles across the ciliopathy spectrum, as sequencing of a diverse cohort identified several missense mutations detrimental to protein function. In addition, in vivo genetic interaction studies indicated that knockdown of KIF7 could exacerbate the phenotype induced by knockdown of other ciliopathy transcripts. Our data show the role of KIF7 in human primary cilia, especially in the Hedgehog pathway through the regulation of GLI targets, and expand the clinical spectrum of ciliopathies. PMID:21552264

Hook2 is involved in the morphogenesis of the primary cilium

PubMed Central

Baron Gaillard, Carole L.; Pallesi-Pocachard, Emilie; Massey-Harroche, Dominique; Richard, Fabrice; Arsanto, Jean-Pierre; Chauvin, Jean-Paul; Lecine, Patrick; Krämer, Helmut; Borg, Jean-Paul; Le Bivic, André

2011-01-01

Primary cilia originate from the centrosome and play essential roles in several cellular, developmental, and pathological processes, but the underlying mechanisms of ciliogenesis are not fully understood. Given the involvement of the adaptor protein Hook2 in centrosomal homeostasis and protein transport to pericentrosomal aggresomes, we explored its role in ciliogenesis. We found that in human retinal epithelial cells, Hook2 localizes at the Golgi apparatus and centrosome/basal body, a strategic partitioning for ciliogenesis. Of importance, Hook2 depletion disrupts ciliogenesis at a stage before the formation of the ciliary vesicle at the distal tip of the mother centriole. Using two hybrid and immunoprecipitation assays and a small interfering RNA strategy, we found that Hook2 interacts with and stabilizes pericentriolar material protein 1 (PCM1), which was reported to be essential for the recruitment of Rab8a, a GTPase that is believed to be crucial for membrane transport to the primary cilium. Of interest, GFP::Rab8a coimmunoprecipitates with endogenous Hook2 and PCM1. Finally, GFP::Rab8a can overcome Hook2 depletion, demonstrating a functional interaction between Hook2 and these two important regulators of ciliogenesis. The data indicate that Hook2 interacts with PCM1 in a complex that also contains Rab8a and regulates a limiting step required for further initiation of ciliogenesis after centriole maturation. PMID:21998199

Bistable synchronization modes in hydrodynamically coupled micro-rotors

NASA Astrophysics Data System (ADS)

Guo, Hanliang; Kanale, Anup; Fuerthauer, Sebastian; Kanso, Eva

2017-11-01

Cilia often beat in synchrony, and they may transition between different synchronization modes in the same cell type. For example, cilia in the mammalian brain ventricles are reported to periodically change their collective beat orientation, providing a cilia-based switch for redirecting the transport of cerebrospinal fluid. Experimental and theoretical evidences suggest that phase coordinations can be achieved solely via hydrodynamical interactions. However, the exact mechanisms responsible for transitioning between various synchronization modes remain illusive. Here, we use a theoretical model where each cilium is represented by a bead moving along a closed trajectory close to a no-slip surface. We investigate the emergent synchronization modes and their stability for various cilia-inspired force profiles. We observe distinct stable synchronization modes between two rotors, including a bistable regime where both in-phase and anti-phase synchronizations are stable. We then extend this analysis to an array of rotors where we demonstrate the dynamical formations of metachronal waves. These findings may help us to understand the origin of synchrony in biological and bio-inspired systems, and the mechanisms underlying transitions between different synchronization modes.

Jump if you can't take the heat: three escape gaits of Paramecium swimming

NASA Astrophysics Data System (ADS)

Baroud, Charles N.; Hamel, Amandine; Fisch, Cathy; Combettes, Laurent; Dupuys-Williams, Pascale

2010-11-01

Paramecium is able to swim at velocities reaching several times its body size per second, by beating its thousands of cilia in an organized fashion. Here we show that Paramecium has in fact three distinct swimming gaits to escape from an aggression in the form of localized heating, depending on the magnitude of the aggression: For a weak agression, normal swimming is sufficient and produces a steady swimming velocity through cilia beating. As the heating amplitude is increased, a higher acceleration and faster swimming are achieved through synchronized beating of the cilia, which later give way to the usual metachronal waves. The synchronized beating yields high initial accelerations but requires the cell to coast through the synchrnized recovery. Finally, escape from a life-threatening agression is achieved by a "jumping" gait which does not rely on the cilia but is achieved from the explosive release of a rod-like organelles in the direction of the hot spot. Measurements through high-speed video explain the role of these rods in defending Paramecium. They also show that the zero-Reynolds number assumption is unverified in most cases.

Optimal ciliary beating patterns

NASA Astrophysics Data System (ADS)

Vilfan, Andrej; Osterman, Natan

2011-11-01

We introduce a measure for energetic efficiency of single or collective biological cilia. We define the efficiency of a single cilium as Q2 / P , where Q is the volume flow rate of the pumped fluid and P is the dissipated power. For ciliary arrays, we define it as (ρQ) 2 / (ρP) , with ρ denoting the surface density of cilia. We then numerically determine the optimal beating patterns according to this criterion. For a single cilium optimization leads to curly, somewhat counterintuitive patterns. But when looking at a densely ciliated surface, the optimal patterns become remarkably similar to what is observed in microorganisms like Paramecium. The optimal beating pattern then consists of a fast effective stroke and a slow sweeping recovery stroke. Metachronal waves lead to a significantly higher efficiency than synchronous beating. Efficiency also increases with an increasing density of cilia up to the point where crowding becomes a problem. We finally relate the pumping efficiency of cilia to the swimming efficiency of a spherical microorganism and show that the experimentally estimated efficiency of Paramecium is surprisingly close to the theoretically possible optimum.

A new index for characterizing micro-bead motion in a flow induced by ciliary beating: Part II, modeling

PubMed Central

Bottier, Mathieu; Peña Fernández, Marta; Pelle, Gabriel; Grotberg, James B.

2017-01-01

Mucociliary clearance is one of the major lines of defense of the human respiratory system. The mucus layer coating the airways is constantly moved along and out of the lung by the activity of motile cilia, expelling at the same time particles trapped in it. The efficiency of the cilia motion can experimentally be assessed by measuring the velocity of micro-beads traveling through the fluid surrounding the cilia. Here we present a mathematical model of the fluid flow and of the micro-beads motion. The coordinated movement of the ciliated edge is represented as a continuous envelope imposing a periodic moving velocity boundary condition on the surrounding fluid. Vanishing velocity and vanishing shear stress boundary conditions are applied to the fluid at a finite distance above the ciliated edge. The flow field is expanded in powers of the amplitude of the individual cilium movement. It is found that the continuous component of the horizontal velocity at the ciliated edge generates a 2D fluid velocity field with a parabolic profile in the vertical direction, in agreement with the experimental measurements. Conversely, we show than this model can be used to extract microscopic properties of the cilia motion by extrapolating the micro-bead velocity measurement at the ciliated edge. Finally, we derive from these measurements a scalar index providing a direct assessment of the cilia beating efficiency. This index can easily be measured in patients without any modification of the current clinical procedures. PMID:28708866

Remodeling Cildb, a popular database for cilia and links for ciliopathies

PubMed Central

2014-01-01

Background New generation technologies in cell and molecular biology generate large amounts of data hard to exploit for individual proteins. This is particularly true for ciliary and centrosomal research. Cildb is a multi–species knowledgebase gathering high throughput studies, which allows advanced searches to identify proteins involved in centrosome, basal body or cilia biogenesis, composition and function. Combined to localization of genetic diseases on human chromosomes given by OMIM links, candidate ciliopathy proteins can be compiled through Cildb searches. Methods Othology between recent versions of the whole proteomes was computed using Inparanoid and ciliary high throughput studies were remapped on these recent versions. Results Due to constant evolution of the ciliary and centrosomal field, Cildb has been recently upgraded twice, with new species whole proteomes and new ciliary studies, and the latter version displays a novel BioMart interface, much more intuitive than the previous ones. Conclusions This already popular database is designed now for easier use and is up to date in regard to high throughput ciliary studies. PMID:25422781

Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes

NASA Technical Reports Server (NTRS)

Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Marshall, Wallace F.

2005-01-01

The important role that cilia and flagella play in human disease creates an urgent need to identify genes involved in ciliary assembly and function. The strong and specific induction of flagellar-coding genes during flagellar regeneration in Chlamydomonas reinhardtii suggests that transcriptional profiling of such cells would reveal new flagella-related genes. We have conducted a genome-wide analysis of RNA transcript levels during flagellar regeneration in Chlamydomonas by using maskless photolithography method-produced DNA oligonucleotide microarrays with unique probe sequences for all exons of the 19,803 predicted genes. This analysis represents previously uncharacterized whole-genome transcriptional activity profiling study in this important model organism. Analysis of strongly induced genes reveals a large set of known flagellar components and also identifies a number of important disease-related proteins as being involved with cilia and flagella, including the zebrafish polycystic kidney genes Qilin, Reptin, and Pontin, as well as the testis-expressed tubby-like protein TULP2.

Vortex arrays and ciliary tangles underlie the feeding-swimming tradeoff in starfish larvae

NASA Astrophysics Data System (ADS)

Gilpin, William; Prakash, Vivek N.; Prakash, Manu

2016-11-01

Many marine invertebrates have larval stages covered in linear arrays of beating cilia, which propel the animal while simultaneously entraining planktonic prey. These bands are strongly conserved across taxa spanning four major superphyla, and they are responsible for the unusual morphologies of many invertebrates. However, few studies have investigated their underlying hydrodynamics. Here, we study the ciliary bands of starfish larvae, and discover a beautiful pattern of slowly-evolving vortices that surrounds the swimming animals. Closer inspection of the bands reveals unusual ciliary "tangles" analogous to topological defects that break-up and re-form as the animal adjusts its swimming stroke. Quantitative experiments and modeling demonstrate that these vortices create a physical tradeoff between feeding and swimming in heterogenous environments, which manifests as distinct flow patterns or "eigenstrokes" representing each behavior-potentially implicating neuronal control of cilia. This quantitative interplay between larval form and hydrodynamic function generalizes to other invertebrates, and illustrates the potential effects of active boundary conditions in other biological and synthetic systems.

Clinical Features and Associated Likelihood of Primary Ciliary Dyskinesia in Children and Adolescents.

PubMed

Leigh, Margaret W; Ferkol, Thomas W; Davis, Stephanie D; Lee, Hye-Seung; Rosenfeld, Margaret; Dell, Sharon D; Sagel, Scott D; Milla, Carlos; Olivier, Kenneth N; Sullivan, Kelli M; Zariwala, Maimoona A; Pittman, Jessica E; Shapiro, Adam J; Carson, Johnny L; Krischer, Jeffrey; Hazucha, Milan J; Knowles, Michael R

2016-08-01

Primary ciliary dyskinesia (PCD), a genetically heterogeneous, recessive disorder of motile cilia, is associated with distinct clinical features. Diagnostic tests, including ultrastructural analysis of cilia, nasal nitric oxide measurements, and molecular testing for mutations in PCD genes, have inherent limitations. To define a statistically valid combination of systematically defined clinical features that strongly associates with PCD in children and adolescents. Investigators at seven North American sites in the Genetic Disorders of Mucociliary Clearance Consortium prospectively and systematically assessed individuals (aged 0-18 yr) referred due to high suspicion for PCD. The investigators defined specific clinical questions for the clinical report form based on expert opinion. Diagnostic testing was performed using standardized protocols and included nasal nitric oxide measurement, ciliary biopsy for ultrastructural analysis of cilia, and molecular genetic testing for PCD-associated genes. Final diagnoses were assigned as "definite PCD" (hallmark ultrastructural defects and/or two mutations in a PCD-associated gene), "probable/possible PCD" (no ultrastructural defect or genetic diagnosis, but compatible clinical features and nasal nitric oxide level in PCD range), and "other diagnosis or undefined." Criteria were developed to define early childhood clinical features on the basis of responses to multiple specific queries. Each defined feature was tested by logistic regression. Sensitivity and specificity analyses were conducted to define the most robust set of clinical features associated with PCD. From 534 participants 18 years of age and younger, 205 were identified as having "definite PCD" (including 164 with two mutations in a PCD-associated gene), 187 were categorized as "other diagnosis or undefined," and 142 were defined as having "probable/possible PCD." Participants with "definite PCD" were compared with the "other diagnosis or undefined" group. Four criteria-defined clinical features were statistically predictive of PCD: laterality defect; unexplained neonatal respiratory distress; early-onset, year-round nasal congestion; and early-onset, year-round wet cough (adjusted odds ratios of 7.7, 6.6, 3.4, and 3.1, respectively). The sensitivity and specificity based on the number of criteria-defined clinical features were four features, 0.21 and 0.99, respectively; three features, 0.50 and 0.96, respectively; and two features, 0.80 and 0.72, respectively. Systematically defined early clinical features could help identify children, including infants, likely to have PCD. Clinical trial registered with ClinicalTrials.gov (NCT00323167).

Morphological studies of the developing human esophageal epithelium.

PubMed

Ménard, D

1995-06-15

This article focusses on the structural development of human esophageal ciliated epithelium. A combination of transmission electron microscopic (TEM), scanning electron microscopic (SEM), radioautographic, and light microscopic (LM) analyses were carried out using intact fetal tissues between 8 and 20 weeks of gestation as well as cultured esophageal explants. Up to the age of 10 weeks, the stratified esophageal epithelium consisted of two longitudinal primary folds. The surface cells were undifferentiated and contained large glycogen aggregates. Between 11 and 16 weeks, the primary folds (now up to four) had developed secondary folds. The thickness of the epithelium drastically increased (123%) in concomittance with a differentiation of surface columnar ciliated cells. These highly specialized surface cells exhibited junctional complexes and well-developed organelles with numerous microvilli interspersed among the cilia. Transverse sections revealed the internal structure of the cilia with a consistent pattern of nine doublet microtubules surrounding a central pair of single microtubules. Freeze-fracture studies illustrated the presence of a ciliary necklace composed of 6 ring-like rows of intramembranous particles. They also revealed the structure of ciliary cell tight junctions consisting of up to nine anastomosing strands (P-face) or complementary grooves (E-face). Ultrastructural studies (LM, TEM, SEM) of the esophageal squamous epithelium obtained after 15 days of culture showed that the newly formed epithelium was similar to adult human epithelium. Finally LM and SEM observations established that the esophagogastric junction was not yet well delineated, consisting of a transitional area composed of a mixture of esophageal ciliated cells and gastric columnar mucous cells.

Dysfunction of intraflagellar transport-A causes hyperphagia-induced obesity and metabolic syndrome

PubMed Central

Jacobs, Damon T.; Silva, Luciane M.; Allard, Bailey A.; Schonfeld, Michael P.; Chatterjee, Anindita; Talbott, George C.

2016-01-01

ABSTRACT Primary cilia extend from the plasma membrane of most vertebrate cells and mediate signaling pathways. Ciliary dysfunction underlies ciliopathies, which are genetic syndromes that manifest multiple clinical features, including renal cystic disease and obesity. THM1 (also termed TTC21B or IFT139) encodes a component of the intraflagellar transport-A complex and mutations in THM1 have been identified in 5% of individuals with ciliopathies. Consistent with this, deletion of murine Thm1 during late embryonic development results in cystic kidney disease. Here, we report that deletion of murine Thm1 during adulthood results in obesity, diabetes, hypertension and fatty liver disease, with gender differences in susceptibility to weight gain and metabolic dysfunction. Pair-feeding of Thm1 conditional knock-out mice relative to control littermates prevented the obesity and related disorders, indicating that hyperphagia caused the obese phenotype. Thm1 ablation resulted in increased localization of adenylyl cyclase III in primary cilia that were shortened, with bulbous distal tips on neurons of the hypothalamic arcuate nucleus, an integrative center for signals that regulate feeding and activity. In pre-obese Thm1 conditional knock-out mice, expression of anorexogenic pro-opiomelanocortin (Pomc) was decreased by 50% in the arcuate nucleus, which likely caused the hyperphagia. Fasting of Thm1 conditional knock-out mice did not alter Pomc nor orexogenic agouti-related neuropeptide (Agrp) expression, suggesting impaired sensing of changes in peripheral signals. Together, these data indicate that the Thm1-mutant ciliary defect diminishes sensitivity to feeding signals, which alters appetite regulation and leads to hyperphagia, obesity and metabolic disease. PMID:27482817

Use of a novel cell adhesion method and digital measurement to show stimulus-dependent variation in somatic and oral ciliary beat frequency in Paramecium.

PubMed

Bell, Wade E; Hallworth, Richard; Wyatt, Todd A; Sisson, Joseph H

2015-01-01

When Paramecium encounters positive stimuli, the membrane hyperpolarizes and ciliary beat frequency increases. We adapted an established immobilization protocol using a biological adhesive and a novel digital analysis system to quantify beat frequency in immobilized Paramecium. Cells showed low mortality and demonstrated beat frequencies consistent with previous studies. Chemoattractant molecules, reduction in external potassium, and posterior stimulation all increased somatic beat frequency. In all cases, the oral groove cilia maintained a higher beat frequency than mid-body cilia, but only oral cilia from cells stimulated with chemoattactants showed an increase from basal levels. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

Corti's organ physiology-based cochlear model: a microelectronic prosthetic implant

NASA Astrophysics Data System (ADS)

Rios, Francisco; Fernandez-Ramos, Raquel; Romero-Sanchez, Jorge; Martin, Jose Francisco

2003-04-01

Corti"s Organ is an Electro-Mechanical transducer that allows the energy coupling between acoustical stimuli and auditory nerve. Although the structure and funtionality of this organ are complex, state of the art models have been currently developed and tested. Cochlea model presented in this paper is based on the theories of Bekesy and others and concerns on the behaviour of auditory system on frequency-place domain and mechanisms of lateral inhibition. At the same time, present state of technology will permit us developing a microsystem that reproduce this phenomena applied to hearing aid prosthesis. Corti"s Organ is composed of more than 20.000 cilia excited by mean of travelling waves. These waves produce relative pressures distributed along the cochlea, exciting an specific number of cilia in a local way. Nonlinear mechanisms of local adaptation to the intensity (external cilia cells) and lateral inhibition (internal cilia cells) allow the selection of very few elements excited. These transmit a very precise intensity and frequency information. These signals are the only ones coupled to the auditory nerve. Distribution of pressure waves matches a quasilogaritmic law due to Cochlea morphology. Microsystem presented in this paper takes Bark"s law as an approximation to this behaviour consisting on grouped arbitrary elements composed of a set of selective coupled exciters (bank of filters according to Patterson"s model).These sets apply the intensity adaptation principles and lateral inhibition. Elements excited during the process generate a bioelectric signal in the same way than cilia cell. A microelectronic solution is presented for the development of an implantable prosthesis device.

Large-scale Synchronization in Carpets of Micro-rotors

NASA Astrophysics Data System (ADS)

Kanale, Anup; Guo, Hanliang; Yan, Wen; Kanso, Eva

2017-11-01

Motile cilia are ubiquitous in nature, and have a critical role in biological locomotion and fluid transport. They often beat in an orchestrated wavelike fashion, and theoretical evidence suggests that this coordinated motion could arise from hydrodynamic interactions. Models based on bead-spring oscillators were used to examine the interaction between pairs of cilia, focusing on in-phase or anti-phase synchrony, while models of hydrodynamically-coupled elastic filaments looked at metachronal coordination in large but finite numbers of interacting cilia. The latter models reproduce metachronal wave coordination, but they are not readily amenable to analysis and parametric studies that highlight the origin of the instabilities that lead to wave propagations and wavelength selection. Here, we use a known model in which each cilium is represented by a rigid sphere moving along a circular trajectory close to a wall, hence the term rotor. The rotor is driven by a cilia-inspired force profile. We generalize this model to a doubly-periodic array of rotors, assuming small distance to the bounding wall, and employ Ewald summation techniques to solve for the flow field. Our goal is to examine the conditions that give rise to stable metachronal waves and their associated wavelength.

Transitions between three swimming gaits in Paramecium escape.

PubMed

Hamel, Amandine; Fisch, Cathy; Combettes, Laurent; Dupuis-Williams, Pascale; Baroud, Charles N

2011-05-03

Paramecium and other protists are able to swim at velocities reaching several times their body size per second by beating their cilia in an organized fashion. The cilia beat in an asymmetric stroke, which breaks the time reversal symmetry of small scale flows. Here we show that Paramecium uses three different swimming gaits to escape from an aggression, applied in the form of a focused laser heating. For a weak aggression, normal swimming is sufficient and produces a steady swimming velocity. As the heating amplitude is increased, a higher acceleration and faster swimming are achieved through synchronized beating of the cilia, which begin by producing oscillating swimming velocities and later give way to the usual gait. Finally, escape from a life-threatening aggression is achieved by a "jumping" gait, which does not rely on the cilia but is achieved through the explosive release of a group of trichocysts in the direction of the hot spot. Measurements through high-speed video explain the role of trichocysts in defending against aggressions while showing unexpected transitions in the swimming of microorganisms. These measurements also demonstrate that Paramecium optimizes its escape pattern by taking advantage of its inertia.

Transitions between three swimming gaits in Paramecium escape

PubMed Central

Hamel, Amandine; Fisch, Cathy; Combettes, Laurent; Dupuis-Williams, Pascale; Baroud, Charles N.

2011-01-01

Paramecium and other protists are able to swim at velocities reaching several times their body size per second by beating their cilia in an organized fashion. The cilia beat in an asymmetric stroke, which breaks the time reversal symmetry of small scale flows. Here we show that Paramecium uses three different swimming gaits to escape from an aggression, applied in the form of a focused laser heating. For a weak aggression, normal swimming is sufficient and produces a steady swimming velocity. As the heating amplitude is increased, a higher acceleration and faster swimming are achieved through synchronized beating of the cilia, which begin by producing oscillating swimming velocities and later give way to the usual gait. Finally, escape from a life-threatening aggression is achieved by a “jumping” gait, which does not rely on the cilia but is achieved through the explosive release of a group of trichocysts in the direction of the hot spot. Measurements through high-speed video explain the role of trichocysts in defending against aggressions while showing unexpected transitions in the swimming of microorganisms. These measurements also demonstrate that Paramecium optimizes its escape pattern by taking advantage of its inertia. PMID:21464291

A mutation in Ccdc39 causes neonatal hydrocephalus with abnormal motile cilia development in mice.

PubMed

Abdelhamed, Zakia; Vuong, Shawn M; Hill, Lauren; Shula, Crystal; Timms, Andrew; Beier, David; Campbell, Kenneth; Mangano, Francesco T; Stottmann, Rolf W; Goto, June

2018-01-09

Pediatric hydrocephalus is characterized by an abnormal accumulation of cerebrospinal fluid (CSF) and is one of the most common congenital brain abnormalities. However, little is known about the molecular and cellular mechanisms regulating CSF flow in the developing brain. Through whole-genome sequencing analysis, we report that a homozygous splice site mutation in coiled-coil domain containing 39 ( Ccdc39 ) is responsible for early postnatal hydrocephalus in the progressive hydrocephal us ( prh ) mouse mutant. Ccdc39 is selectively expressed in embryonic choroid plexus and ependymal cells on the medial wall of the forebrain ventricle, and the protein is localized to the axoneme of motile cilia. The Ccdc39 prh/prh ependymal cells develop shorter cilia with disorganized microtubules lacking the axonemal inner arm dynein. Using high-speed video microscopy, we show that an orchestrated ependymal ciliary beating pattern controls unidirectional CSF flow on the ventricular surface, which generates bulk CSF flow in the developing brain. Collectively, our data provide the first evidence for involvement of Ccdc39 in hydrocephalus and suggest that the proper development of medial wall ependymal cilia is crucial for normal mouse brain development. © 2018. Published by The Company of Biologists Ltd.

The Centrioles, Centrosomes, Basal Bodies, and Cilia of Drosophila melanogaster

PubMed Central

Lattao, Ramona; Kovács, Levente; Glover, David M.

2017-01-01

Centrioles play a key role in the development of the fly. They are needed for the correct formation of centrosomes, the organelles at the poles of the spindle that can persist as microtubule organizing centers (MTOCs) into interphase. The ability to nucleate cytoplasmic microtubules (MTs) is a property of the surrounding pericentriolar material (PCM). The centriole has a dual life, existing not only as the core of the centrosome but also as the basal body, the structure that templates the formation of cilia and flagellae. Thus the structure and functions of the centriole, the centrosome, and the basal body have an impact upon many aspects of development and physiology that can readily be modeled in Drosophila. Centrosomes are essential to give organization to the rapidly increasing numbers of nuclei in the syncytial embryo and for the spatially precise execution of cell division in numerous tissues, particularly during male meiosis. Although mitotic cell cycles can take place in the absence of centrosomes, this is an error-prone process that opens up the fly to developmental defects and the potential of tumor formation. Here, we review the structure and functions of the centriole, the centrosome, and the basal body in different tissues and cultured cells of Drosophila melanogaster, highlighting their contributions to different aspects of development and cell division. PMID:28476861

Wnt/PCP Instructions for Cilia in Left-Right Asymmetry.

PubMed

Wu, Jun; Mlodzik, Marek

2017-03-13

Wnt-Frizzled/planar cell polarity (PCP) signaling establishes cell orientation within the epithelial plane, but whether Wnts are instructive or permissive is debated. Reporting in Developmental Cell, Minegishi et al. (2017) uncover an instructive link from Wnt5a/b gradients to PCP-factor-regulated polarized cilia positioning that is essential to mouse embryo left-right asymmetry establishment. Copyright © 2017. Published by Elsevier Inc.

Use of optical tweezers to probe epithelial mechanosensation

NASA Astrophysics Data System (ADS)

Resnick, Andrew

2010-01-01

Cellular mechanosensation mechanisms have been implicated in a variety of disease states. Specifically in renal tubules, the primary cilium and associated mechanosensitive ion channels are hypothesized to play a role in water and salt homeostasis, with relevant disease states including polycystic kidney disease and hypertension. Previous experiments investigating ciliary-mediated cellular mechanosensation have used either fluid flow chambers or micropipetting to elicit a biological response. The interpretation of these experiments in terms of the ``ciliary hypothesis'' has been difficult due the spatially distributed nature of the mechanical disturbance-several competing hypotheses regarding possible roles of primary cilium, glycocalyx, microvilli, cell junctions, and actin cytoskeleton exist. I report initial data using optical tweezers to manipulate individual primary cilia in an attempt to elicit a mechanotransduction response-specifically, the release of intracellular calcium. The advantage of using laser tweezers over previous work is that the applied disturbance is highly localized. I find that stimulation of a primary cilium elicits a response, while stimulation of the apical surface membrane does not. These results lend support to the hypothesis that the primary cilium mediates transduction of mechanical strain into a biochemical response in renal epithelia.

Dysfunction of intraflagellar transport-A causes hyperphagia-induced obesity and metabolic syndrome.

PubMed

Jacobs, Damon T; Silva, Luciane M; Allard, Bailey A; Schonfeld, Michael P; Chatterjee, Anindita; Talbott, George C; Beier, David R; Tran, Pamela V

2016-07-01

Primary cilia extend from the plasma membrane of most vertebrate cells and mediate signaling pathways. Ciliary dysfunction underlies ciliopathies, which are genetic syndromes that manifest multiple clinical features, including renal cystic disease and obesity. THM1 (also termed TTC21B or IFT139) encodes a component of the intraflagellar transport-A complex and mutations in THM1 have been identified in 5% of individuals with ciliopathies. Consistent with this, deletion of murine Thm1 during late embryonic development results in cystic kidney disease. Here, we report that deletion of murine Thm1 during adulthood results in obesity, diabetes, hypertension and fatty liver disease, with gender differences in susceptibility to weight gain and metabolic dysfunction. Pair-feeding of Thm1 conditional knock-out mice relative to control littermates prevented the obesity and related disorders, indicating that hyperphagia caused the obese phenotype. Thm1 ablation resulted in increased localization of adenylyl cyclase III in primary cilia that were shortened, with bulbous distal tips on neurons of the hypothalamic arcuate nucleus, an integrative center for signals that regulate feeding and activity. In pre-obese Thm1 conditional knock-out mice, expression of anorexogenic pro-opiomelanocortin (Pomc) was decreased by 50% in the arcuate nucleus, which likely caused the hyperphagia. Fasting of Thm1 conditional knock-out mice did not alter Pomc nor orexogenic agouti-related neuropeptide (Agrp) expression, suggesting impaired sensing of changes in peripheral signals. Together, these data indicate that the Thm1-mutant ciliary defect diminishes sensitivity to feeding signals, which alters appetite regulation and leads to hyperphagia, obesity and metabolic disease. © 2016. Published by The Company of Biologists Ltd.

IFT88 influences chondrocyte actin organization and biomechanics.

PubMed

Wang, Z; Wann, A K T; Thompson, C L; Hassen, A; Wang, W; Knight, M M

2016-03-01

Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88(orpk)). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. IFT88(orpk) cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88(orpk) cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88(orpk) cells. Following membrane blebbing, IFT88(orpk) cells exhibited slower reformation of the actin cortex. IFT88(orpk) cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Laterality Defects Other Than Situs Inversus Totalis in Primary Ciliary Dyskinesia

PubMed Central

Davis, Stephanie D.; Ferkol, Thomas; Dell, Sharon D.; Rosenfeld, Margaret; Olivier, Kenneth N.; Sagel, Scott D.; Milla, Carlos; Zariwala, Maimoona A.; Wolf, Whitney; Carson, Johnny L.; Hazucha, Milan J.; Burns, Kimberlie; Robinson, Blair; Knowles, Michael R.; Leigh, Margaret W.

2014-01-01

BACKGROUND: Motile cilia dysfunction causes primary ciliary dyskinesia (PCD), situs inversus totalis (SI), and a spectrum of laterality defects, yet the prevalence of laterality defects other than SI in PCD has not been prospectively studied. METHODS: In this prospective study, participants with suspected PCD were referred to our multisite consortium. We measured nasal nitric oxide (nNO) level, examined cilia with electron microscopy, and analyzed PCD-causing gene mutations. Situs was classified as (1) situs solitus (SS), (2) SI, or (3) situs ambiguus (SA), including heterotaxy. Participants with hallmark electron microscopic defects, biallelic gene mutations, or both were considered to have classic PCD. RESULTS: Of 767 participants (median age, 8.1 years, range, 0.1-58 years), classic PCD was defined in 305, including 143 (46.9%), 125 (41.0%), and 37 (12.1%) with SS, SI, and SA, respectively. A spectrum of laterality defects was identified with classic PCD, including 2.6% and 2.3% with SA plus complex or simple cardiac defects, respectively; 4.6% with SA but no cardiac defect; and 2.6% with an isolated possible laterality defect. Participants with SA and classic PCD had a higher prevalence of PCD-associated respiratory symptoms vs SA control participants (year-round wet cough, P < .001; year-round nasal congestion, P = .015; neonatal respiratory distress, P = .009; digital clubbing, P = .021) and lower nNO levels (median, 12 nL/min vs 252 nL/min; P < .001). CONCLUSIONS: At least 12.1% of patients with classic PCD have SA and laterality defects ranging from classic heterotaxy to subtle laterality defects. Specific clinical features of PCD and low nNO levels help to identify PCD in patients with laterality defects. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT00323167; URL: www.clinicaltrials.gov PMID:24577564

Chloral hydrate alters the organization of the ciliary basal apparatus and cell organelles in sea urchin embryos

NASA Technical Reports Server (NTRS)

Chakrabarti, A.; Schatten, H.; Mitchell, K. D.; Crosser, M.; Taylor, M.

1998-01-01

The mitotic inhibitor, chloral hydrate, induces ciliary loss in the early embryo phase of Lytechinus pictus. It causes a breakdown of cilia at the junction of the cilium and the basal body known as the basal plate. This leaves the plasma membrane temporarily unsealed. The basal apparatus accessory structures, consisting of the basal body, basal foot, basal foot cap, striated side arm, and striated rootlet, are either misaligned or disintegrated by treatment with chloral hydrate. Furthermore, microtubules which are associated with the basal apparatus are disassembled. Mitochondria accumulate at the base of cilia - underneath the plasma membrane - and show alterations in their structural organization. The accumulation of mitochondria is observed in 40% of all electron micrograph sections while 60% show the areas mostly devoid of mitochondria. The microvilli surrounding a cilium and striated rootlet remain intact in the presence of chloral hydrate. These results suggest that deciliation in early sea urchin embryos by chloral hydrate is caused by combined effects on the ciliary membrane and on microtubules in the cilia. Furthermore, it is suggested that chloral hydrate can serve as a tool to explore the cytoskeletal mechanisms that are involved in cilia motility in the developing sea urchin embryo.

The spatiotemporal organization of cilia activity drives multiscale circular flows of mucus in reconstituted human bronchial epithelium

NASA Astrophysics Data System (ADS)

Loiseau, Etienne; Gras, Delphine; Chanez, Pascal; Viallat, Annie

2017-11-01

Chronic respiratory diseases affect hundreds of millions of people worldwide. The bronchial epithelium is the first barrier to protect the respiratory tract via an innate mechanism called mucociliary clearance. It consists in the active transport of a sticky fluid, the mucus, via a myriad of cilia at the epithelial surface of the airways. The mucus traps inhaled pathogens and the protective role of the mucociliary clearance relies on the ability of the cilia to self-organize and coordinate their beating to transport the mucus over the full bronchial tree till its elimination through swallowing or expectoration. Despite a rich corpus of clinical studies, chronic respiratory diseases remain poorly understood and quantitative biophysical studies are still missing. Here we will present the physical mechanisms underlying the mucociliary transport. We will show how the cilia self-organize during the ciliogenesis and how the coordination of their beating direction leads to the formation of fluid flow patterns at the macroscopic scale. Finally, we will discuss the role of long range hydrodynamics interactions in this intricate coupled system. ANR MUCOCIL project, Grant ANR-13-BSV5-0015 and European Union's Seventh Framework Programme (FP7/2007-2013) under REA Grant agreement n. PCOFUND-GA-2013-609102.

Mutations in the Katnb1 gene cause left-right asymmetry and heart defects.

PubMed

Furtado, Milena B; Merriner, D Jo; Berger, Silke; Rhodes, Danielle; Jamsai, Duangporn; O'Bryan, Moira K

2017-12-01

The microtubule-severing protein complex katanin is composed two subunits, the ATPase subunit, KATNA1, and the noncatalytic regulatory subunit, KATNB1. Recently, the Katnb1 gene has been linked to infertility, regulation of centriole and cilia formation in fish and mammals, as well as neocortical brain development. KATNB1 protein is expressed in germ cells in humans and mouse, mitotic/meiotic spindles and cilia, although the full expression pattern of the Katnb1 gene has not been described. Using a knockin-knockout mouse model of Katnb1 dysfunction we demonstrate that Katnb1 is ubiquitously expressed during embryonic development, although a stronger expression is seen in the crown cells of the gastrulation organizer, the murine node. Furthermore, null and hypomorphic Katnb1 gene mutations show a novel correlation between Katnb1 dysregulation and the development of impaired left-right signaling, including cardiac malformations. Katanin function is a critical regulator of heart development in mice. These findings are potentially relevant to human cardiac development. Developmental Dynamics 246:1027-1035, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Cryoelectron tomography of radial spokes in cilia and flagella

PubMed Central

Pigino, Gaia; Bui, Khanh Huy; Maheshwari, Aditi; Lupetti, Pietro; Diener, Dennis

2011-01-01

Radial spokes (RSs) are ubiquitous components in the 9 + 2 axoneme thought to be mechanochemical transducers involved in local control of dynein-driven microtubule sliding. They are composed of >23 polypeptides, whose interactions and placement must be deciphered to understand RS function. In this paper, we show the detailed three-dimensional (3D) structure of RS in situ in Chlamydomonas reinhardtii flagella and Tetrahymena thermophila cilia that we obtained using cryoelectron tomography (cryo-ET). We clarify similarities and differences between the three spoke species, RS1, RS2, and RS3, in T. thermophila and in C. reinhardtii and show that part of RS3 is conserved in C. reinhardtii, which only has two species of complete RSs. By analyzing C. reinhardtii mutants, we identified the specific location of subsets of RS proteins (RSPs). Our 3D reconstructions show a twofold symmetry, suggesting that fully assembled RSs are produced by dimerization. Based on our cryo-ET data, we propose models of subdomain organization within the RS as well as interactions between RSPs and with other axonemal components. PMID:22065640

Vortex arrays and ciliary tangles underlie the feeding-swimming trade-off in starfish larvae

NASA Astrophysics Data System (ADS)

Gilpin, William; Prakash, Vivek N.; Prakash, Manu

2017-04-01

Many marine invertebrates have larval stages covered in linear arrays of beating cilia, which propel the animal while simultaneously entraining planktonic prey. These bands are strongly conserved across taxa spanning four major superphyla, and they are responsible for the unusual morphologies of many invertebrate larvae. However, few studies have investigated their underlying hydrodynamics. Here, we study the ciliary bands of starfish larvae, and discover a beautiful pattern of slowly evolving vortices that surrounds the swimming animals. Closer inspection of the bands reveals unusual ciliary `tangles' analogous to topological defects that break up and re-form as the animal adjusts its swimming stroke. Quantitative experiments and modelling demonstrate that these vortices create a physical trade-off between feeding and swimming in heterogeneous environments, which manifests as distinct flow patterns or `eigenstrokes' representing each behaviour--potentially implicating neuronal control of cilia. This quantitative interplay between larval form and hydrodynamic function may generalize to other invertebrates with ciliary bands, and illustrates the potential effects of active boundary conditions in other biological and synthetic systems.

Small organelle, big responsibility: the role of centrosomes in development and disease.

PubMed

Chavali, Pavithra L; Pütz, Monika; Gergely, Fanni

2014-09-05

The centrosome, a key microtubule organizing centre, is composed of centrioles, embedded in a protein-rich matrix. Centrosomes control the internal spatial organization of somatic cells, and as such contribute to cell division, cell polarity and migration. Upon exiting the cell cycle, most cell types in the human body convert their centrioles into basal bodies, which drive the assembly of primary cilia, involved in sensing and signal transduction at the cell surface. Centrosomal genes are targeted by mutations in numerous human developmental disorders, ranging from diseases exclusively affecting brain development, through global growth failure syndromes to diverse pathologies associated with ciliary malfunction. Despite our much-improved understanding of centrosome function in cellular processes, we know remarkably little of its role in the organismal context, especially in mammals. In this review, we examine how centrosome dysfunction impacts on complex physiological processes and speculate on the challenges we face when applying knowledge generated from in vitro and in vivo model systems to human development. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

DOE PAGES

Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut; ...

2014-12-02

During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut

During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

TRPP2 ion channels: Critical regulators of organ morphogenesis in health and disease.

PubMed

Busch, Tilman; Köttgen, Michael; Hofherr, Alexis

2017-09-01

Ion channels control the membrane potential and mediate transport of ions across membranes. Archetypical physiological functions of ion channels include processes such as regulation of neuronal excitability, muscle contraction, or transepithelial ion transport. In that regard, transient receptor potential ion channel polycystin 2 (TRPP2) is remarkable, because it controls complex morphogenetic processes such as the establishment of properly shaped epithelial tubules and left-right-asymmetry of organs. The fascinating question of how an ion channel regulates morphogenesis has since captivated the attention of scientists in different disciplines. Four loosely connected key insights on different levels of biological complexity ranging from protein to whole organism have framed our understanding of TRPP2 physiology: 1) TRPP2 is a non-selective cation channel; 2) TRPP2 is part of a receptor-ion channel complex; 3) TRPP2 localizes to primary cilia; and 4) TRPP2 is required for organ morphogenesis. In this review, we will discuss the current knowledge in these key areas and highlight some of the challenges ahead. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

TMEM237 Is Mutated in Individuals with a Joubert Syndrome Related Disorder and Expands the Role of the TMEM Family at the Ciliary Transition Zone

PubMed Central

Huang, Lijia; Szymanska, Katarzyna; Jensen, Victor L.; Janecke, Andreas R.; Innes, A. Micheil; Davis, Erica E.; Frosk, Patrick; Li, Chunmei; Willer, Jason R.; Chodirker, Bernard N.; Greenberg, Cheryl R.; McLeod, D. Ross; Bernier, Francois P.; Chudley, Albert E.; Müller, Thomas; Shboul, Mohammad; Logan, Clare V.; Loucks, Catrina M.; Beaulieu, Chandree L.; Bowie, Rachel V.; Bell, Sandra M.; Adkins, Jonathan; Zuniga, Freddi I.; Ross, Kevin D.; Wang, Jian; Ban, Matthew R.; Becker, Christian; Nürnberg, Peter; Douglas, Stuart; Craft, Cheryl M.; Akimenko, Marie-Andree; Hegele, Robert A.; Ober, Carole; Utermann, Gerd; Bolz, Hanno J.; Bulman, Dennis E.; Katsanis, Nicholas; Blacque, Oliver E.; Doherty, Dan; Parboosingh, Jillian S.; Leroux, Michel R.; Johnson, Colin A.; Boycott, Kym M.

2011-01-01

Joubert syndrome related disorders (JSRDs) have broad but variable phenotypic overlap with other ciliopathies. The molecular etiology of this overlap is unclear but probably arises from disrupting common functional module components within primary cilia. To identify additional module elements associated with JSRDs, we performed homozygosity mapping followed by next-generation sequencing (NGS) and uncovered mutations in TMEM237 (previously known as ALS2CR4). We show that loss of the mammalian TMEM237, which localizes to the ciliary transition zone (TZ), results in defective ciliogenesis and deregulation of Wnt signaling. Furthermore, disruption of Danio rerio (zebrafish) tmem237 expression produces gastrulation defects consistent with ciliary dysfunction, and Caenorhabditis elegans jbts-14 genetically interacts with nphp-4, encoding another TZ protein, to control basal body-TZ anchoring to the membrane and ciliogenesis. Both mammalian and C. elegans TMEM237/JBTS-14 require RPGRIP1L/MKS5 for proper TZ localization, and we demonstrate additional functional interactions between C. elegans JBTS-14 and MKS-2/TMEM216, MKSR-1/B9D1, and MKSR-2/B9D2. Collectively, our findings integrate TMEM237/JBTS-14 in a complex interaction network of TZ-associated proteins and reveal a growing contribution of a TZ functional module to the spectrum of ciliopathy phenotypes. PMID:22152675

Clinical Features and Associated Likelihood of Primary Ciliary Dyskinesia in Children and Adolescents

PubMed Central

Ferkol, Thomas W.; Davis, Stephanie D.; Lee, Hye-Seung; Rosenfeld, Margaret; Dell, Sharon D.; Sagel, Scott D.; Milla, Carlos; Olivier, Kenneth N.; Sullivan, Kelli M.; Zariwala, Maimoona A.; Pittman, Jessica E.; Shapiro, Adam J.; Carson, Johnny L.; Krischer, Jeffrey; Hazucha, Milan J.

2016-01-01

Rationale: Primary ciliary dyskinesia (PCD), a genetically heterogeneous, recessive disorder of motile cilia, is associated with distinct clinical features. Diagnostic tests, including ultrastructural analysis of cilia, nasal nitric oxide measurements, and molecular testing for mutations in PCD genes, have inherent limitations. Objectives: To define a statistically valid combination of systematically defined clinical features that strongly associates with PCD in children and adolescents. Methods: Investigators at seven North American sites in the Genetic Disorders of Mucociliary Clearance Consortium prospectively and systematically assessed individuals (aged 0–18 yr) referred due to high suspicion for PCD. The investigators defined specific clinical questions for the clinical report form based on expert opinion. Diagnostic testing was performed using standardized protocols and included nasal nitric oxide measurement, ciliary biopsy for ultrastructural analysis of cilia, and molecular genetic testing for PCD-associated genes. Final diagnoses were assigned as “definite PCD” (hallmark ultrastructural defects and/or two mutations in a PCD-associated gene), “probable/possible PCD” (no ultrastructural defect or genetic diagnosis, but compatible clinical features and nasal nitric oxide level in PCD range), and “other diagnosis or undefined.” Criteria were developed to define early childhood clinical features on the basis of responses to multiple specific queries. Each defined feature was tested by logistic regression. Sensitivity and specificity analyses were conducted to define the most robust set of clinical features associated with PCD. Measurements and Main Results: From 534 participants 18 years of age and younger, 205 were identified as having “definite PCD” (including 164 with two mutations in a PCD-associated gene), 187 were categorized as “other diagnosis or undefined,” and 142 were defined as having “probable/possible PCD.” Participants with “definite PCD” were compared with the “other diagnosis or undefined” group. Four criteria-defined clinical features were statistically predictive of PCD: laterality defect; unexplained neonatal respiratory distress; early-onset, year-round nasal congestion; and early-onset, year-round wet cough (adjusted odds ratios of 7.7, 6.6, 3.4, and 3.1, respectively). The sensitivity and specificity based on the number of criteria-defined clinical features were four features, 0.21 and 0.99, respectively; three features, 0.50 and 0.96, respectively; and two features, 0.80 and 0.72, respectively. Conclusions: Systematically defined early clinical features could help identify children, including infants, likely to have PCD. Clinical trial registered with ClinicalTrials.gov (NCT00323167). PMID:27070726

Effect of mycotoxins on in vitro movement of tracheal cilia from one-day-old chicks.

PubMed

Jesenská, Z; Bernát, D

1994-01-01

The effect of 11 mycotoxins on the ciliary movement of tracheal epithelium from one-day-old chicks in vitro was examined. Sterigmatocystin and diacetoxyscirpenol were most ciliostatically active in vitro; the ciliostatic effect was observed after 2 d if the amount concentration was 30 micrograms/L. In contrast, patulin stopped the movement of cilia after 2 d only if its concentration was 20 mg/L.

Flies without centrioles.

PubMed

Basto, Renata; Lau, Joyce; Vinogradova, Tatiana; Gardiol, Alejandra; Woods, C Geoffrey; Khodjakov, Alexey; Raff, Jordan W

2006-06-30

Centrioles and centrosomes have an important role in animal cell organization, but it is uncertain to what extent they are essential for animal development. The Drosophila protein DSas-4 is related to the human microcephaly protein CenpJ and the C. elegans centriolar protein Sas-4. We show that DSas-4 is essential for centriole replication in flies. DSas-4 mutants start to lose centrioles during embryonic development, and, by third-instar larval stages, no centrioles or centrosomes are detectable. Mitotic spindle assembly is slow in mutant cells, and approximately 30% of the asymmetric divisions of larval neuroblasts are abnormal. Nevertheless, mutant flies develop with near normal timing into morphologically normal adults. These flies, however, have no cilia or flagella and die shortly after birth because their sensory neurons lack cilia. Thus, centrioles are essential for the formation of centrosomes, cilia, and flagella, but, remarkably, they are not essential for most aspects of Drosophila development.

Mechanistic basis of otolith formation during teleost inner ear development

PubMed Central

Wu, David; Freund, Jonathan B.; Fraser, Scott E.; Vermot, Julien

2011-01-01

Otoliths, which are connected to stereociliary bundles in the inner ear, serve as inertial sensors for balance. In teleostei, otolith development is critically dependant on flow forces generated by beating cilia; however, the mechanism by which flow controls otolith formation remains unclear. Here, we have developed a non-invasive flow probe using optical tweezers and a viscous flow model in order to demonstrate how the observed hydrodynamics influence otolith assembly. We show that rotational flow stirs and suppresses precursor agglomeration in the core of the cilia-driven vortex. The velocity field correlates with the shape of the otolith and we provide evidence that hydrodynamics is actively involved in controlling otolith morphogenesis. An implication of this hydrodynamic effect is that otolith self-assembly is mediated by the balance between Brownian motion and cilia-driven flow. More generally, this flow feature highlights an alternative biological strategy for controlling particle localization in solution. PMID:21316594

Primary ciliary dyskinesia. Recent advances in diagnostics, genetics, and characterization of clinical disease.

PubMed

Knowles, Michael R; Daniels, Leigh Anne; Davis, Stephanie D; Zariwala, Maimoona A; Leigh, Margaret W

2013-10-15

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia that leads to oto-sino-pulmonary diseases and organ laterality defects in approximately 50% of cases. The estimated incidence of PCD is approximately 1 per 15,000 births, but the prevalence of PCD is difficult to determine, primarily because of limitations in diagnostic methods that focus on testing ciliary ultrastructure and function. Diagnostic capabilities have recently benefitted from (1) documentation of low nasal nitric oxide production in PCD and (2) discovery of biallelic mutations in multiple PCD-causing genes. The use of these complementary diagnostic approaches shows that at least 30% of patients with PCD have normal ciliary ultrastructure. More accurate identification of patients with PCD has also allowed definition of a strong clinical phenotype, which includes neonatal respiratory distress in >80% of cases, daily nasal congestion and wet cough starting soon after birth, and early development of recurrent/chronic middle-ear and sinus disease. Recent studies, using advanced imaging and pulmonary physiologic assessments, clearly demonstrate early onset of lung disease in PCD, with abnormal air flow mechanics by age 6-8 years that is similar to cystic fibrosis, and age-dependent onset of bronchiectasis. The treatment of PCD is not standardized, and there are no validated PCD-specific therapies. Most patients with PCD receive suboptimal management, which should include airway clearance, regular surveillance of pulmonary function and respiratory microbiology, and use of antibiotics targeted to pathogens. The PCD Foundation is developing a network of clinical centers, which should improve diagnosis and management of PCD.

Defects in neural stem cell proliferation and olfaction in Chd7 deficient mice indicate a mechanism for hyposmia in human CHARGE syndrome

PubMed Central

Layman, W.S.; McEwen, D.P.; Beyer, L.A.; Lalani, S.R.; Fernbach, S.D.; Oh, E.; Swaroop, A.; Hegg, C.C.; Raphael, Y.; Martens, J.R.; Martin, D.M.

2009-01-01

Mutations in CHD7, a chromodomain gene, are present in a majority of individuals with CHARGE syndrome, a multiple anomaly disorder characterized by ocular Coloboma, Heart defects, Atresia of the choanae, Retarded growth and development, Genital hypoplasia and Ear anomalies. The clinical features of CHARGE syndrome are highly variable and incompletely penetrant. Olfactory dysfunction is a common feature in CHARGE syndrome and has been potentially linked to primary olfactory bulb defects, but no data confirming this mechanistic link have been reported. On the basis of these observations, we hypothesized that loss of Chd7 disrupts mammalian olfactory tissue development and function. We found severe defects in olfaction in individuals with CHD7 mutations and CHARGE, and loss of odor evoked electro-olfactogram responses in Chd7 deficient mice, suggesting reduced olfaction is due to a dysfunctional olfactory epithelium. Chd7 expression was high in basal olfactory epithelial neural stem cells and down-regulated in mature olfactory sensory neurons. We observed smaller olfactory bulbs, reduced olfactory sensory neurons, and disorganized epithelial ultrastructure in Chd7 mutant mice, despite apparently normal functional cilia and sustentacular cells. Significant reductions in the proliferation of neural stem cells and regeneration of olfactory sensory neurons in the mature Chd7Gt/+ olfactory epithelium indicate critical roles for Chd7 in regulating neurogenesis. These studies provide evidence that mammalian olfactory dysfunction due to Chd7 haploinsufficiency is linked to primary defects in olfactory neural stem cell proliferation and may influence olfactory bulb development. PMID:19279158

Primary Ciliary Dyskinesia. Recent Advances in Diagnostics, Genetics, and Characterization of Clinical Disease

PubMed Central

Daniels, Leigh Anne; Davis, Stephanie D.; Zariwala, Maimoona A.; Leigh, Margaret W.

2013-01-01

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia that leads to oto-sino-pulmonary diseases and organ laterality defects in approximately 50% of cases. The estimated incidence of PCD is approximately 1 per 15,000 births, but the prevalence of PCD is difficult to determine, primarily because of limitations in diagnostic methods that focus on testing ciliary ultrastructure and function. Diagnostic capabilities have recently benefitted from (1) documentation of low nasal nitric oxide production in PCD and (2) discovery of biallelic mutations in multiple PCD-causing genes. The use of these complementary diagnostic approaches shows that at least 30% of patients with PCD have normal ciliary ultrastructure. More accurate identification of patients with PCD has also allowed definition of a strong clinical phenotype, which includes neonatal respiratory distress in >80% of cases, daily nasal congestion and wet cough starting soon after birth, and early development of recurrent/chronic middle-ear and sinus disease. Recent studies, using advanced imaging and pulmonary physiologic assessments, clearly demonstrate early onset of lung disease in PCD, with abnormal air flow mechanics by age 6–8 years that is similar to cystic fibrosis, and age-dependent onset of bronchiectasis. The treatment of PCD is not standardized, and there are no validated PCD-specific therapies. Most patients with PCD receive suboptimal management, which should include airway clearance, regular surveillance of pulmonary function and respiratory microbiology, and use of antibiotics targeted to pathogens. The PCD Foundation is developing a network of clinical centers, which should improve diagnosis and management of PCD. PMID:23796196

Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome

PubMed Central

Alby, Caroline; Piquand, Kevin; Huber, Céline; Megarbané, André; Ichkou, Amale; Legendre, Marine; Pelluard, Fanny; Encha-Ravazi, Ferechté; Abi-Tayeh, Georges; Bessières, Bettina; El Chehadeh-Djebbar, Salima; Laurent, Nicole; Faivre, Laurence; Sztriha, László; Zombor, Melinda; Szabó, Hajnalka; Failler, Marion; Garfa-Traore, Meriem; Bole, Christine; Nitschké, Patrick; Nizon, Mathilde; Elkhartoufi, Nadia; Clerget-Darpoux, Françoise; Munnich, Arnold; Lyonnet, Stanislas; Vekemans, Michel; Saunier, Sophie; Cormier-Daire, Valérie; Attié-Bitach, Tania; Thomas, Sophie

2015-01-01

KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies. PMID:26166481

Effects of Heparin and Lisofylline on Pulmonary Function Following Smoke Inhalation Injury in an Ovine Model

DTIC Science & Technology

2002-03-01

25 mg/kg intravenously). One radiopaque sheath introducer, through which a pulmonary artery flotation catheter was placed, was inserted into an...the tracheobronchoepithelial damage score was as follows: 0, normal; 1, some loss of cilia, loss of apical epithelium; 2, marked attenuation of epi...limited to the left apical lobe be- cause bronchoalveolar lavage was done in the other lobes. The lung parenchymal damage score was as follows: 0, normal; 1

The Centrioles, Centrosomes, Basal Bodies, and Cilia of Drosophila melanogaster.

PubMed

Lattao, Ramona; Kovács, Levente; Glover, David M

2017-05-01

Centrioles play a key role in the development of the fly. They are needed for the correct formation of centrosomes, the organelles at the poles of the spindle that can persist as microtubule organizing centers (MTOCs) into interphase. The ability to nucleate cytoplasmic microtubules (MTs) is a property of the surrounding pericentriolar material (PCM). The centriole has a dual life, existing not only as the core of the centrosome but also as the basal body, the structure that templates the formation of cilia and flagellae. Thus the structure and functions of the centriole, the centrosome, and the basal body have an impact upon many aspects of development and physiology that can readily be modeled in Drosophila Centrosomes are essential to give organization to the rapidly increasing numbers of nuclei in the syncytial embryo and for the spatially precise execution of cell division in numerous tissues, particularly during male meiosis. Although mitotic cell cycles can take place in the absence of centrosomes, this is an error-prone process that opens up the fly to developmental defects and the potential of tumor formation. Here, we review the structure and functions of the centriole, the centrosome, and the basal body in different tissues and cultured cells of Drosophila melanogaster , highlighting their contributions to different aspects of development and cell division. Copyright © 2017 Lattao et al.

Disruption of IFT Complex A Causes Cystic Kidneys without Mitotic Spindle Misorientation

PubMed Central

Jonassen, Julie A.; SanAgustin, Jovenal; Baker, Stephen P.

2012-01-01

Intraflagellar transport (IFT) complexes A and B build and maintain primary cilia. In the mouse, kidney-specific or hypomorphic mutant alleles of IFT complex B genes cause polycystic kidneys, but the influence of IFT complex A proteins on renal development is not well understood. In the present study, we found that HoxB7-Cre–driven deletion of the complex A gene Ift140 from collecting ducts disrupted, but did not completely prevent, cilia assembly. Mutant kidneys developed collecting duct cysts by postnatal day 5, with rapid cystic expansion and renal dysfunction by day 15 and little remaining parenchymal tissue by day 20. In contrast to many models of polycystic kidney disease, precystic Ift140-deleted collecting ducts showed normal centrosomal positioning and no misorientation of the mitotic spindle axis, suggesting that disruption of oriented cell division is not a prerequisite to cyst formation in these kidneys. Precystic collecting ducts had an increased mitotic index, suggesting that cell proliferation may drive cyst expansion even with normal orientation of the mitotic spindle. In addition, we observed significant increases in expression of canonical Wnt pathway genes and mediators of Hedgehog and tissue fibrosis in highly cystic, but not precystic, kidneys. Taken together, these studies indicate that loss of Ift140 causes pronounced renal cystic disease and suggest that abnormalities in several different pathways may influence cyst progression. PMID:22282595

ULTRASTRUCTURAL ORGANIZATION OF CILIA AND BASAL BODIES OF THE EPITHELIUM OF THE CHOROID PLEXUS IN THE CHICK EMBRYO

PubMed Central

Doolin, Paul F.; Birge, Wesley J.

1966-01-01

Ultrastructural studies were performed on normal and abnormal cilia and basal bodies associated with the choroidal epithelium of the chick embryo. Tissues were prepared in each of several fixatives including: 1% osmium tetroxide, in both phosphate and veronal acetate buffers; 2% glutaraldehyde, followed by postfixation in osmium tetroxide; 1% potassium permanganate in veronal acetate buffer. Normal cilia display the typical pattern of 9 peripheral doublets and 2 central fibers, as well as a system of 9 secondary fibers. The latter show distinct interconnections between peripheral and central fibers. Supernumerary fibers were found to occur in certain abnormal cilia. The basal body is complex, bearing 9 transitional fibers at the distal end and numerous cross-striated rootlets at the proximal end. The distal end of the basal body is delimited by a basal plate of moderate density. The tubular cylinder consists of 9 triple fibers. The C subfibers end at the basal plate, whereas subfibers A and B continue into the shaft of the cilium. The 9 transitional fibers radiate out from the distal end of the basal body, ending in bulblike terminal enlargements which are closely associated with the cell membrane in the area of the basal cup. One or 2 prominent basal feet project laterally from the basal body. These structures characteristically show several dense cross-bands and, on occasion, are found associated with microtubules. PMID:5335827

Morphogenesis of the node and notochord: the cellular basis for the establishment and maintenance of left-right asymmetry in the mouse.

PubMed

Lee, Jeffrey D; Anderson, Kathryn V

2008-12-01

Establishment of left-right asymmetry in the mouse embryo depends on leftward laminar fluid flow in the node, which initiates a signaling cascade that is confined to the left side of the embryo. Leftward fluid flow depends on two cellular processes: motility of the cilia that generate the flow and morphogenesis of the node, the structure where the cilia reside. Here, we provide an overview of the current understanding and unresolved questions about the regulation of ciliary motility and node structure. Analysis of mouse mutants has shown that the motile cilia must have a specific structure and length, and that they must point posteriorly to generate the necessary leftward fluid flow. However, the precise structure of the motile cilia is not clear and the mechanisms that position cilia on node cells have not been defined. The mouse node is a teardrop-shaped pit at the distal tip of the early embryo, but the morphogenetic events that create the mature node from cells derived from the primitive streak are only beginning to be characterized. Recent live imaging experiments support earlier scanning electron microscopy (SEM) studies and show that node assembly is a multi-step process in which clusters of node precursors appear on the embryo surface as overlying endoderm cells are removed. We present additional SEM and confocal microscopy studies that help define the transition stages during node morphogenesis. After the initiation of left-sided signaling, the notochordal plate, which is contiguous with the node, generates a barrier at the embryonic midline that restricts the cascade of gene expression to the left side of the embryo. The field is now poised to dissect the genetic and cellular mechanisms that create and organize the specialized cells of the node and midline that are essential for left-right asymmetry. (c) 2008 Wiley-Liss, Inc.

Conservation of Planar Polarity Pathway Function Across the Animal Kingdom.

PubMed

Hale, Rosalind; Strutt, David

2015-01-01

Planar polarity is a well-studied phenomenon resulting in the directional coordination of cells in the plane of a tissue. In invertebrates and vertebrates, planar polarity is established and maintained by the largely independent core and Fat/Dachsous/Four-jointed (Ft-Ds-Fj) pathways. Loss of function of these pathways can result in a wide range of developmental or cellular defects, including failure of gastrulation and problems with placement and function of cilia. This review discusses the conservation of these pathways across the animal kingdom. The lack of vital core pathway components in basal metazoans suggests that the core planar polarity pathway evolved shortly after, but not necessarily alongside, the emergence of multicellularity.

Gain-of-function mutations in the gene encoding the tyrosine phosphatase SHP2 induce hydrocephalus in a catalytically dependent manner.

PubMed

Zheng, Hong; Yu, Wen-Mei; Waclaw, Ronald R; Kontaridis, Maria I; Neel, Benjamin G; Qu, Cheng-Kui

2018-03-20

Catalytically activating mutations in Ptpn11 , which encodes the protein tyrosine phosphatase SHP2, cause 50% of Noonan syndrome (NS) cases, whereas inactivating mutations in Ptpn11 are responsible for nearly all cases of the similar, but distinct, developmental disorder Noonan syndrome with multiple lentigines (NSML; formerly called LEOPARD syndrome). However, both types of disease mutations are gain-of-function mutations because they cause SHP2 to constitutively adopt an open conformation. We found that the catalytic activity of SHP2 was required for the pathogenic effects of gain-of-function, disease-associated mutations on the development of hydrocephalus in the mouse. Targeted pan-neuronal knockin of a Ptpn11 allele encoding the active SHP2 E76K mutant resulted in hydrocephalus due to aberrant development of ependymal cells and their cilia. These pathogenic effects of the E76K mutation were suppressed by the additional mutation C459S, which abolished the catalytic activity of SHP2. Moreover, ependymal cells in NSML mice bearing the inactive SHP2 mutant Y279C were also unaffected. Mechanistically, the SHP2 E76K mutant induced developmental defects in ependymal cells by enhancing dephosphorylation and inhibition of the transcription activator STAT3. Whereas STAT3 activity was reduced in Ptpn11 E76K/+ cells, the activities of the kinases ERK and AKT were enhanced, and neural cell-specific Stat3 knockout mice also manifested developmental defects in ependymal cells and cilia. These genetic and biochemical data demonstrate a catalytic-dependent role of SHP2 gain-of-function disease mutants in the pathogenesis of hydrocephalus. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Toolkit for the Automated Characterization of Optical Trapping Forces on Microscopic Particles

NASA Astrophysics Data System (ADS)

Glaser, Joseph; Hoeprich, David; Resnick, Andrew

2014-03-01

Optical traps have been in use in microbiological studies for the past 40 years to obtain noninvasive control of microscopic particles. However, the magnitude of the applied forces is often unknown. Therefore, we have developed an automated data acquisition and processing system which characterizes trap properties for known particle geometries. Extensive experiments and measurements utilizing well-characterized objects were performed and compared to literature to confirm the system's performance. This system will enable the future analysis of a trapped primary cilium, a slender rod-shaped organelle with aspect ratio L/R >30, where `L' is the cilium length and `R' the cilium diameter. The trapping of cilia is of primary importance, as it will lead to the precise measurements of mechanical properties of the organelle and its significance to the epithelial cell. Support from the National Institutes of Health, 1R15DK092716 is gratefully acknowledged.

Dishevelled links basal body docking and orientation in ciliated epithelial cells

PubMed Central

Vladar, Eszter K.; Axelrod, Jeffrey D.

2014-01-01

Some epithelia contain cells with multiple, motile cilia that beat in a concerted fashion. New tools and experimental systems have facilitated molecular studies of cilium biogenesis and of the coordinated planar polarization of cilia that leads to their concerted motility. Recent, elegant work by Park and colleagues, using embryonic frog epidermis, demonstrates that Dishevelled (Dvl), a key regulator of both the Wnt/β-catenin and Planar Cell Polarity (PCP) pathways, controls both the docking and planar polarization of ciliary basal bodies. PMID:18819800

Molecular characterization of the apical organ of the anthozoan Nematostella vectensis

PubMed Central

Sinigaglia, Chiara; Busengdal, Henriette; Lerner, Avi; Oliveri, Paola; Rentzsch, Fabian

2015-01-01

Apical organs are sensory structures present in many marine invertebrate larvae where they are considered to be involved in their settlement, metamorphosis and locomotion. In bilaterians they are characterised by a tuft of long cilia and receptor cells and they are associated with groups of neurons, but their relatively low morphological complexity and dispersed phylogenetic distribution have left their evolutionary relationship unresolved. Moreover, since apical organs are not present in the standard model organisms, their development and function are not well understood. To provide a foundation for a better understanding of this structure we have characterised the molecular composition of the apical organ of the sea anemone Nematostella vectensis. In a microarray-based comparison of the gene expression profiles of planulae with either a wildtype or an experimentally expanded apical organ, we identified 78 evolutionarily conserved genes, which are predominantly or specifically expressed in the apical organ of Nematostella. This gene set comprises signalling molecules, transcription factors, structural and metabolic genes. The majority of these genes, including several conserved, but previously uncharacterized ones, are potentially involved in different aspects of the development or function of the long cilia of the apical organ. To demonstrate the utility of this gene set for comparative analyses, we further analysed the expression of a subset of previously uncharacterized putative orthologs in sea urchin larvae and detected expression for twelve out of eighteen of them in the apical domain. Our study provides a molecular characterization of the apical organ of Nematostella and represents an informative tool for future studies addressing the development, function and evolutionary history of apical organ cells. PMID:25478911

The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions

PubMed Central

Merchant, Sabeeha S.; Prochnik, Simon E.; Vallon, Olivier; Harris, Elizabeth H.; Karpowicz, Steven J.; Witman, George B.; Terry, Astrid; Salamov, Asaf; Fritz-Laylin, Lillian K.; Maréchal-Drouard, Laurence; Marshall, Wallace F.; Qu, Liang-Hu; Nelson, David R.; Sanderfoot, Anton A.; Spalding, Martin H.; Kapitonov, Vladimir V.; Ren, Qinghu; Ferris, Patrick; Lindquist, Erika; Shapiro, Harris; Lucas, Susan M.; Grimwood, Jane; Schmutz, Jeremy; Cardol, Pierre; Cerutti, Heriberto; Chanfreau, Guillaume; Chen, Chun-Long; Cognat, Valérie; Croft, Martin T.; Dent, Rachel; Dutcher, Susan; Fernández, Emilio; Ferris, Patrick; Fukuzawa, Hideya; González-Ballester, David; González-Halphen, Diego; Hallmann, Armin; Hanikenne, Marc; Hippler, Michael; Inwood, William; Jabbari, Kamel; Kalanon, Ming; Kuras, Richard; Lefebvre, Paul A.; Lemaire, Stéphane D.; Lobanov, Alexey V.; Lohr, Martin; Manuell, Andrea; Meier, Iris; Mets, Laurens; Mittag, Maria; Mittelmeier, Telsa; Moroney, James V.; Moseley, Jeffrey; Napoli, Carolyn; Nedelcu, Aurora M.; Niyogi, Krishna; Novoselov, Sergey V.; Paulsen, Ian T.; Pazour, Greg; Purton, Saul; Ral, Jean-Philippe; Riaño-Pachón, Diego Mauricio; Riekhof, Wayne; Rymarquis, Linda; Schroda, Michael; Stern, David; Umen, James; Willows, Robert; Wilson, Nedra; Zimmer, Sara Lana; Allmer, Jens; Balk, Janneke; Bisova, Katerina; Chen, Chong-Jian; Elias, Marek; Gendler, Karla; Hauser, Charles; Lamb, Mary Rose; Ledford, Heidi; Long, Joanne C.; Minagawa, Jun; Page, M. Dudley; Pan, Junmin; Pootakham, Wirulda; Roje, Sanja; Rose, Annkatrin; Stahlberg, Eric; Terauchi, Aimee M.; Yang, Pinfen; Ball, Steven; Bowler, Chris; Dieckmann, Carol L.; Gladyshev, Vadim N.; Green, Pamela; Jorgensen, Richard; Mayfield, Stephen; Mueller-Roeber, Bernd; Rajamani, Sathish; Sayre, Richard T.; Brokstein, Peter; Dubchak, Inna; Goodstein, David; Hornick, Leila; Huang, Y. Wayne; Jhaveri, Jinal; Luo, Yigong; Martínez, Diego; Ngau, Wing Chi Abby; Otillar, Bobby; Poliakov, Alexander; Porter, Aaron; Szajkowski, Lukasz; Werner, Gregory; Zhou, Kemin; Grigoriev, Igor V.; Rokhsar, Daniel S.; Grossman, Arthur R.

2010-01-01

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella. PMID:17932292

Spot Surface Labeling of Magnetic Microbeads and Application in Biological Force Measurements

NASA Astrophysics Data System (ADS)

Estes, Ashley; O'Brien, E. Tim; Hill, David; Superfine, Richard

2006-11-01

Biological force measurements on single molecules and macromolecular structures often use microbeads for the application of force. These techniques are often complicated by multiple attachments and nonspecific binding. In one set of experiments, we are applying a magnetic force microscope that allows us to pull on magnetic beads attached to ciliated human bronchial epithelial cells. These experiments provide a means to measure the stall force of cilia and understand how cilia propel fluids. However, because we are using beads with diameters of one and 2.8 microns, and the diameter of human airway cilia is approximately 200 nm, we cannot be assured that the bead is bound to a single cilium. To address this, we have developed a sputter coating technique to block the biotin binding capability of the streptavidin labeled bead over its entire surface except for a small spot. These beads may also have applications in other biological experiments such as DNA force experiments in which binding of a single target to an individual bead is critical.

Effect of Cilia Beat Frequency on Muco-ciliary Clearance

PubMed Central

Sedaghat, M.H.; Shahmardan, M.M.; Norouzi, M.; Heydari, M.

2016-01-01

Background: The airway surface liquid (ASL), which is a fluid layer coating the interior epithelial surface of the bronchi and bronchiolesis, plays an important defensive role against foreign particles and chemicals entering lungs. Objective: Numerical investigation has been employed to solve two-layer model consisting of mucus layer as a viscoelastic fluid and periciliary liquid layer as a Newtonian fluid to study the effects of cilia beat frequency (CBF) at various amounts of mucus properties on muco-ciliary transport problem. Methods: Hybrid finite difference-lattice Boltzmann-method (FB-LBM) has been used to solve the momentum equations and to simulate cilia forces, and also the PCL-mucus interface more accurately, immersed boundary method (IBM) has been employed. The main contribution of the current study is to use an Oldroyd-B model as the constitutive equation of mucus. Results: Our results show that increasing CBF and decreasing mucus viscosity ratio have great effects on mucus flow, but the effect of viscosity ratio is more significant. The results also illustrate that the relation between cilia beat frequency and mean mucus velocity is almost linear and it has similar behavior at different values of viscosity ratio. Conclusion: Numerical investigation based on hybrid IB-FD-LBM has been used to study the effect of CBF at various mounts of mucus viscosity ratio on the muco-ciliary clearance. The results showed that the effect of viscosity ratio on the muco-ciliary transport process is more significant compared with CBF. PMID:28144596

Mutations in the Gene Encoding IFT Dynein Complex Component WDR34 Cause Jeune Asphyxiating Thoracic Dystrophy

PubMed Central

Schmidts, Miriam; Vodopiutz, Julia; Christou-Savina, Sonia; Cortés, Claudio R.; McInerney-Leo, Aideen M.; Emes, Richard D.; Arts, Heleen H.; Tüysüz, Beyhan; D’Silva, Jason; Leo, Paul J.; Giles, Tom C.; Oud, Machteld M.; Harris, Jessica A.; Koopmans, Marije; Marshall, Mhairi; Elçioglu, Nursel; Kuechler, Alma; Bockenhauer, Detlef; Moore, Anthony T.; Wilson, Louise C.; Janecke, Andreas R.; Hurles, Matthew E.; Emmet, Warren; Gardiner, Brooke; Streubel, Berthold; Dopita, Belinda; Zankl, Andreas; Kayserili, Hülya; Scambler, Peter J.; Brown, Matthew A.; Beales, Philip L.; Wicking, Carol; Duncan, Emma L.; Mitchison, Hannah M.

2013-01-01

Bidirectional (anterograde and retrograde) motor-based intraflagellar transport (IFT) governs cargo transport and delivery processes that are essential for primary cilia growth and maintenance and for hedgehog signaling functions. The IFT dynein-2 motor complex that regulates ciliary retrograde protein transport contains a heavy chain dynein ATPase/motor subunit, DYNC2H1, along with other less well functionally defined subunits. Deficiency of IFT proteins, including DYNC2H1, underlies a spectrum of skeletal ciliopathies. Here, by using exome sequencing and a targeted next-generation sequencing panel, we identified a total of 11 mutations in WDR34 in 9 families with the clinical diagnosis of Jeune syndrome (asphyxiating thoracic dystrophy). WDR34 encodes a WD40 repeat-containing protein orthologous to Chlamydomonas FAP133, a dynein intermediate chain associated with the retrograde intraflagellar transport motor. Three-dimensional protein modeling suggests that the identified mutations all affect residues critical for WDR34 protein-protein interactions. We find that WDR34 concentrates around the centrioles and basal bodies in mammalian cells, also showing axonemal staining. WDR34 coimmunoprecipitates with the dynein-1 light chain DYNLL1 in vitro, and mining of proteomics data suggests that WDR34 could represent a previously unrecognized link between the cytoplasmic dynein-1 and IFT dynein-2 motors. Together, these data show that WDR34 is critical for ciliary functions essential to normal development and survival, most probably as a previously unrecognized component of the mammalian dynein-IFT machinery. PMID:24183451

Functional Architecture of the Outer Arm Dynein Conformational Switch*

PubMed Central

King, Stephen M.; Patel-King, Ramila S.

2012-01-01

Dynein light chain 1 (LC1/DNAL1) is one of the most highly conserved components of ciliary axonemal outer arm dyneins, and it associates with both a heavy chain motor unit and tubulin located within the A-tubule of the axonemal outer doublet microtubules. In a variety of model systems, lack of LC1 or expression of mutant forms leads to profound defects in ciliary motility, including the failure of the hydrodynamic coupling needed for ciliary metachronal synchrony, random stalling during the power/recovery stroke transition, an aberrant response to imposed viscous load, and in some cases partial failure of motor assembly. These phenotypes have led to the proposal that LC1 acts as part of a mechanical switch to control motor function in response to alterations in axonemal curvature. Here we have used NMR chemical shift mapping to define the regions perturbed by a series of mutations in the C-terminal domain that yield a range of phenotypic effects on motility. In addition, we have identified the subdomain of LC1 involved in binding microtubules and characterized the consequences of an Asn → Ser alteration within the terminal leucine-rich repeat that in humans causes primary ciliary dyskinesia. Together, these data define a series of functional subdomains within LC1 and allow us to propose a structural model for the organization of the dynein heavy chain-LC1-microtubule ternary complex that is required for the coordinated activity of dynein motors in cilia. PMID:22157010

Functional Surface of the golden mussel's foot: morphology, structures and the role of cilia on underwater adhesion.

PubMed

Andrade, Gabriela Rabelo; de Araújo, João Locke Ferreira; Nakamura Filho, Arnaldo; Guañabens, Anna Carolina Paganini; Carvalho, Marcela David de; Cardoso, Antônio Valadão

2015-09-01

In this study we characterized the surface morphology and ultrastructure of the foot of the golden mussel, Limnoperna fortunei (Dunker, 1857), relating its characteristics to the attaching mechanisms of this mollusk. The observation of the foot of this bivalve reveals the presence of micro-scaled cilia with a unique shape, which has a narrowing at its end. This characteristic was associated to the capacity for underwater adhesion to substrates through the employment of van der Waals forces, resembling the adhesion phenomenon of the gecko foot. The temporary attachment during locomotion by means of the foot to substrates was observed to be strong even on smooth surfaces, like glass, or hydrophobic waxy surfaces. Comparing TEM and light microscopy results it was possible to associate the mucous secretions and secreting cells found along the tissues to the production of the byssus inside the groove on the ventral portion of the foot. Not only our experiments, but also the state of the art allowed us to discard the involvement of secretions produced in the foot of the mussel to the temporary adhesion. Through SEM images it was possible to build a virtual three-dimensional model where total foot surface was measured for the estimated calculation of van der Waals forces. Also, some theoretical analysis and considerations have been made concerning the characteristics of the functional surface of L. fortunei foot. Copyright © 2015 Elsevier B.V. All rights reserved.

Tissue-engineered tracheal reconstruction using three-dimensionally printed artificial tracheal graft: preliminary report.

PubMed

Chang, Jae Won; Park, Su A; Park, Ju-Kyeong; Choi, Jae Won; Kim, Yoo-Suk; Shin, Yoo Seob; Kim, Chul-Ho

2014-06-01

Three-dimensional printing has come into the spotlight in the realm of tissue engineering. We intended to evaluate the plausibility of 3D-printed (3DP) scaffold coated with mesenchymal stem cells (MSCs) seeded in fibrin for the repair of partial tracheal defects. MSCs from rabbit bone marrow were expanded and cultured. A half-pipe-shaped 3DP polycaprolactone scaffold was coated with the MSCs seeded in fibrin. The half-pipe tracheal graft was implanted on a 10 × 10-mm artificial tracheal defect in four rabbits. Four and eight weeks after the operation, the reconstructed sites were evaluated bronchoscopically, radiologically, histologically, and functionally. None of the four rabbits showed any sign of respiratory distress. Endoscopic examination and computed tomography showed successful reconstruction of trachea without any collapse or blockage. The replaced tracheas were completely covered with regenerated respiratory mucosa. Histologic analysis showed that the implanted 3DP tracheal grafts were successfully integrated with the adjacent trachea without disruption or granulation tissue formation. Neo-cartilage formation inside the implanted graft was sufficient to maintain the patency of the reconstructed trachea. Scanning electron microscope examination confirmed the regeneration of the cilia, and beating frequency of regenerated cilia was not different from those of the normal adjacent mucosa. The shape and function of reconstructed trachea using 3DP scaffold coated with MSCs seeded in fibrin were restored successfully without any graft rejection. Copyright © 2014 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

ZMYND10 stabilizes intermediate chain proteins in the cytoplasmic pre-assembly of dynein arms.

PubMed

Cho, Kyeong Jee; Noh, Shin Hye; Han, Soo Min; Choi, Won-Il; Kim, Hye-Youn; Yu, Seyoung; Lee, Joon Suk; Rim, John Hoon; Lee, Min Goo; Hildebrandt, Friedhelm; Gee, Heon Yung

2018-03-01

Zinc finger MYND-type-containing 10 (ZMYND10), a cytoplasmic protein expressed in ciliated cells, causes primary ciliary dyskinesia (PCD) when mutated; however, its function is poorly understood. Therefore, in this study, we examined the roles of ZMYND10 using Zmynd10-/-mice exhibiting typical PCD phenotypes, including hydrocephalus and laterality defects. In these mutants, morphology, the number of motile cilia, and the 9+2 axoneme structure were normal; however, inner and outer dynein arms (IDA and ODA, respectively) were absent. ZMYND10 interacted with ODA components and proteins, including LRRC6, DYX1C1, and C21ORF59, implicated in the cytoplasmic pre-assembly of DAs, whose levels were significantly reduced in Zmynd10-/-mice. LRRC6 and DNAI1 were more stable when co-expressed with ZYMND10 than when expressed alone. DNAI2, which did not interact with ZMYND10, was not stabilized by co-expression with ZMYND10 alone, but was stabilized by co-expression with DNAI1 and ZMYND10, suggesting that ZMYND10 stabilized DNAI1, which subsequently stabilized DNAI2. Together, these results demonstrated that ZMYND10 regulated the early stage of DA cytoplasmic pre-assembly by stabilizing DNAI1.

Measurement of ciliary beat frequency using ultra-high resolution optical coherence tomography

NASA Astrophysics Data System (ADS)

Chen, Jason J.; Jing, Joseph C.; Su, Erica; Badger, Christopher; Coughlan, Carolyn A.; Chen, Zhongping; Wong, Brian J. F.

2016-02-01

Ciliated epithelial cells populate up to 80% of the surface area of the human airway and are responsible for mucociliary transport, which is the key protective mechanism that provides the first line of defense in the respiratory tract. Cilia beat in a rhythmic pattern and may be easily affected by allergens, pollutants, and pathogens, altering ciliary beat frequency (CBF) subsequently. Diseases including cystic fibrosis, chronic obstructive pulmonary disease, and primary ciliary dyskinesia may also decrease CBF. CBF is therefore a critical component of respiratory health. The current clinical method of measuring CBF is phase-contrast microscopy, which involves a tissue biopsy obtained via brushing of the nasal cavity. While this method is minimally invasive, the tissue sample must be oriented to display its profile view, making the visualization of a single layer of cilia challenging. In addition, the conventional method requires subjective analysis of CBF, e.g., manually counting by visual inspection. On the contrary, optical coherence tomography (OCT) has been used to study the retina in ophthalmology as well as vasculature in cardiology, and offers higher resolution than conventional computed tomography and magnetic resonance imaging. Based on this technology, our lab specifically developed an ultra-high resolution OCT system to image the microstructure of the ciliated epithelial cells. Doppler analysis was also performed to determine CBF. Lastly, we also developed a program that utilizes fast Fourier transform to determine CBF under phase-contrast microscopy, providing a more objective method compared to the current method.

Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome.

PubMed

Alby, Caroline; Piquand, Kevin; Huber, Céline; Megarbané, André; Ichkou, Amale; Legendre, Marine; Pelluard, Fanny; Encha-Ravazi, Ferechté; Abi-Tayeh, Georges; Bessières, Bettina; El Chehadeh-Djebbar, Salima; Laurent, Nicole; Faivre, Laurence; Sztriha, László; Zombor, Melinda; Szabó, Hajnalka; Failler, Marion; Garfa-Traore, Meriem; Bole, Christine; Nitschké, Patrick; Nizon, Mathilde; Elkhartoufi, Nadia; Clerget-Darpoux, Françoise; Munnich, Arnold; Lyonnet, Stanislas; Vekemans, Michel; Saunier, Sophie; Cormier-Daire, Valérie; Attié-Bitach, Tania; Thomas, Sophie

2015-08-06

KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

SNX27 Deletion Causes Hydrocephalus by Impairing Ependymal Cell Differentiation and Ciliogenesis

PubMed Central

Zhou, Ying; Wang, Jian; Tseng, I-Chu; Huang, Timothy; Zhao, Yingjun; Zheng, Qiuyang; Gao, Yue; Luo, Hong; Zhang, Xian; Bu, Guojun; Hong, Wanjin

2016-01-01

Hydrocephalus is a brain disorder derived from CSF accumulation due to defects in CSF clearance. Although dysfunctional apical cilia in the ependymal cell layer are causal to the onset of hydrocephalus, mechanisms underlying proper ependymal cell differentiation are largely unclear. SNX27 is a trafficking component required for normal brain function and was shown previously to suppress γ-secretase-dependent amyloid precursor protein and Notch cleavage. However, it was unclear how SNX27-dependent γ-secretase inhibition could contribute to brain development and pathophysiology. Here, we describe and characterize an Snx27-deleted mouse model for the ependymal layer defects of deciliation and hydrocephalus. SNX27 deficiency results in reductions in ependymal cells and cilia density, as well as severe postnatal hydrocephalus. Inhibition of Notch intracellular domain signaling with γ-secretase inhibitors reversed ependymal cells/cilia loss and dilation of lateral ventricles in Snx27-deficient mice, giving strong indication that Snx27 deletion triggers defects in ependymal layer formation and ciliogenesis through Notch hyperactivation. Together, these results suggest that SNX27 is essential for ependymal cell differentiation and ciliogenesis, and its deletion can promote hydrocephalus pathogenesis. SIGNIFICANCE STATEMENT Down's syndrome (DS) in humans and mouse models has been shown previously to confer a high risk for the development of pathological hydrocephalus. Because we have previously described SNX27 as a component that is consistently downregulated in DS, we present here a robust Snx27-deleted mouse model that produces hydrocephalus and associated ciliary defects with complete penetrance. In addition, we find that γ-secretase/Notch modulation may be a candidate drug target in SNX27-associated hydrocephalus such as that observed in DS. Based on these findings, we anticipate that future study will determine whether modulation of a SNX27/Notch/γ-secretase pathway can also be of therapeutic interest to congenital hydrocephalus. PMID:27974614

SNX27 Deletion Causes Hydrocephalus by Impairing Ependymal Cell Differentiation and Ciliogenesis.

PubMed

Wang, Xin; Zhou, Ying; Wang, Jian; Tseng, I-Chu; Huang, Timothy; Zhao, Yingjun; Zheng, Qiuyang; Gao, Yue; Luo, Hong; Zhang, Xian; Bu, Guojun; Hong, Wanjin; Xu, Huaxi

2016-12-14

Hydrocephalus is a brain disorder derived from CSF accumulation due to defects in CSF clearance. Although dysfunctional apical cilia in the ependymal cell layer are causal to the onset of hydrocephalus, mechanisms underlying proper ependymal cell differentiation are largely unclear. SNX27 is a trafficking component required for normal brain function and was shown previously to suppress γ-secretase-dependent amyloid precursor protein and Notch cleavage. However, it was unclear how SNX27-dependent γ-secretase inhibition could contribute to brain development and pathophysiology. Here, we describe and characterize an Snx27-deleted mouse model for the ependymal layer defects of deciliation and hydrocephalus. SNX27 deficiency results in reductions in ependymal cells and cilia density, as well as severe postnatal hydrocephalus. Inhibition of Notch intracellular domain signaling with γ-secretase inhibitors reversed ependymal cells/cilia loss and dilation of lateral ventricles in Snx27-deficient mice, giving strong indication that Snx27 deletion triggers defects in ependymal layer formation and ciliogenesis through Notch hyperactivation. Together, these results suggest that SNX27 is essential for ependymal cell differentiation and ciliogenesis, and its deletion can promote hydrocephalus pathogenesis. Down's syndrome (DS) in humans and mouse models has been shown previously to confer a high risk for the development of pathological hydrocephalus. Because we have previously described SNX27 as a component that is consistently downregulated in DS, we present here a robust Snx27-deleted mouse model that produces hydrocephalus and associated ciliary defects with complete penetrance. In addition, we find that γ-secretase/Notch modulation may be a candidate drug target in SNX27-associated hydrocephalus such as that observed in DS. Based on these findings, we anticipate that future study will determine whether modulation of a SNX27/Notch/γ-secretase pathway can also be of therapeutic interest to congenital hydrocephalus. Copyright © 2016 the authors 0270-6474/16/3612586-12$15.00/0.

KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome.

PubMed

Sanders, Anna A W M; de Vrieze, Erik; Alazami, Anas M; Alzahrani, Fatema; Malarkey, Erik B; Sorusch, Nasrin; Tebbe, Lars; Kuhns, Stefanie; van Dam, Teunis J P; Alhashem, Amal; Tabarki, Brahim; Lu, Qianhao; Lambacher, Nils J; Kennedy, Julie E; Bowie, Rachel V; Hetterschijt, Lisette; van Beersum, Sylvia; van Reeuwijk, Jeroen; Boldt, Karsten; Kremer, Hannie; Kesterson, Robert A; Monies, Dorota; Abouelhoda, Mohamed; Roepman, Ronald; Huynen, Martijn H; Ueffing, Marius; Russell, Rob B; Wolfrum, Uwe; Yoder, Bradley K; van Wijk, Erwin; Alkuraya, Fowzan S; Blacque, Oliver E

2015-12-29

Joubert syndrome (JBTS) and related disorders are defined by cerebellar malformation (molar tooth sign), together with neurological symptoms of variable expressivity. The ciliary basis of Joubert syndrome related disorders frequently extends the phenotype to tissues such as the eye, kidney, skeleton and craniofacial structures. Using autozygome and exome analyses, we identified a null mutation in KIAA0556 in a multiplex consanguineous family with hallmark features of mild Joubert syndrome. Patient-derived fibroblasts displayed reduced ciliogenesis potential and abnormally elongated cilia. Investigation of disease pathophysiology revealed that Kiaa0556 (-/-) null mice possess a Joubert syndrome-associated brain-restricted phenotype. Functional studies in Caenorhabditis elegans nematodes and cultured human cells support a conserved ciliary role for KIAA0556 linked to microtubule regulation. First, nematode KIAA0556 is expressed almost exclusively in ciliated cells, and the worm and human KIAA0556 proteins are enriched at the ciliary base. Second, C. elegans KIAA0056 regulates ciliary A-tubule number and genetically interacts with an ARL13B (JBTS8) orthologue to control cilium integrity. Third, human KIAA0556 binds to microtubules in vitro and appears to stabilise microtubule networks when overexpressed. Finally, human KIAA0556 biochemically interacts with ciliary proteins and p60/p80 katanins. The latter form a microtubule-severing enzyme complex that regulates microtubule dynamics as well as ciliary functions. We have identified KIAA0556 as a novel microtubule-associated ciliary base protein mutated in Joubert syndrome. Consistent with the mild patient phenotype, our nematode, mice and human cell data support the notion that KIAA0556 has a relatively subtle and variable cilia-related function, which we propose is related to microtubule regulation.

Ovarian Brenner tumour: a morphologic and immunohistochemical analysis suggesting an origin from fallopian tube epithelium.

PubMed

Kuhn, Elisabetta; Ayhan, Ayse; Shih, Ie-Ming; Seidman, Jeffrey D; Kurman, Robert J

2013-12-01

Brenner tumours (BTs), like other epithelial ovarian tumours, are thought to develop from the ovarian surface epithelium. We hypothesised that BTs arise from transitional metaplasia near the tuboperitoneal junction which, when embedded in the ovary as Walthard cell nests, may progress to BTs. The aim of this study was to validate this hypothesis by a morphologic and immunohistochemical (IHC) analysis. The IHC analysis revealed that fallopian tube secretory cells, transitional metaplasia, Walthard cell nests and the epithelial component of BTs shared a similar IHC profile, consistently expressing AKR1C3 (an enzyme involved in androgen biosynthesis) and androgen receptor, but not calretinin. The tumour stromal cells that immediately surrounded the epithelial nests showed strong expression of calretinin, inhibin and steroidogenic factor 1 (markers of steroidogenic cells) in the majority of BTs. Using a highly sensitive immunofluorescent staining method, we detected small groups of cilia in transitional metaplasia and Walthard cell nests, multifocal stretches of cilia and/or ciliated vacuoles in benign BTs and well-developed cilia in atypical proliferative BTs. Our findings suggest a tubal origin of BTs through transitional metaplasia and Walthard cell nests, based on their anatomic proximity, similar IHC profile and the presence of cilia. In addition, we hypothesise a role of androgenic stimulation in the pathogenesis of BT, based on the IHC staining pattern of calretinin, inhibin and steroidogenic factor 1 expressed in the luteinised stromal cells surrounding the epithelial nests of the tumours, and AKR1C3 and androgen receptor expressed in both the epithelial and stromal components. Copyright © 2013 Elsevier Ltd. All rights reserved.

TTC25 Deficiency Results in Defects of the Outer Dynein Arm Docking Machinery and Primary Ciliary Dyskinesia with Left-Right Body Asymmetry Randomization.

PubMed

Wallmeier, Julia; Shiratori, Hidetaka; Dougherty, Gerard W; Edelbusch, Christine; Hjeij, Rim; Loges, Niki T; Menchen, Tabea; Olbrich, Heike; Pennekamp, Petra; Raidt, Johanna; Werner, Claudius; Minegishi, Katsura; Shinohara, Kyosuke; Asai, Yasuko; Takaoka, Katsuyoshi; Lee, Chanjae; Griese, Matthias; Memari, Yasin; Durbin, Richard; Kolb-Kokocinski, Anja; Sauer, Sascha; Wallingford, John B; Hamada, Hiroshi; Omran, Heymut

2016-08-04

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Active fluid mixing with magnetic microactuators for capture of salmonella

NASA Astrophysics Data System (ADS)

Hanasoge, S.; Owen, D.; Ballard, M.; Mills, Z.; Xu, J.; Erickson, M.; Hesketh, P. J.; Alexeev, A.

2016-05-01

Detection of low concentrations of bacteria in food samples is a challenging process. Key to this process is the separation of the target from the food matrix. We demonstrate magnetic beads and magnetic micro-cilia based microfluidic mixing and capture, which are particularly useful for pre-concentrating the target. The first method we demonstrate makes use of magnetic microbeads held on to NiFe discs on the surface of the substrate. These beads are rotated around the magnetic discs by rotating the external magnetic field. The second method we demonstrate shows the use of cilia which extends into the fluid and is manipulated by a rotating external field. Magnetic micro-features were fabricated by evaporating NiFe alloy at room temperature, on to patterned photoresist. The high magnetic permeability of NiFe allows for maximum magnetic force on the features. The magnetic features were actuated using an external rotating magnet up to frequencies of 50Hz. We demonstrate active mixing produced by the microbeads and the cilia in a microchannel. Also, we demonstrate the capture of target species in a sample using microbeads.

Chirality of the cytoskeleton in the origins of cellular asymmetry

PubMed Central

2016-01-01

Self-assembly of two important components of the cytoskeleton of eukaryotic cells, actin microfilaments and microtubules (MTs) results in polar filaments of one chirality. As is true for bacterial flagella, in actin microfilaments, screw direction is important for assembly processes and motility. For MTs, polar orientation within the cell is paramount. The alignment of these elements in the cell cytoplasm gives rise to emergent properties, including the potential for cell differentiation and specialization. Complex MTs with a characteristic chirality are found in basal bodies and centrioles; this chirality is preserved in cilia. In motile cilia, it is reflected in the direction of the effective stroke. The positioning of the basal body or cilia on the cell surface depends on polarity proteins. In evolution, survival depends on global polarity information relayed to the cell in part by orientation of the MT and actin filament cytoskeletons and the chirality of the basal body to determine left and right coordinates within a defined anterior–posterior cell and tissue axis. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’. PMID:27821520

SHIP2 Regulates Lumen Generation, Cell Division, and Ciliogenesis through the Control of Basolateral to Apical Lumen Localization of Aurora A and HEF 1.

PubMed

Hamze-Komaiha, Ola; Sarr, Sokavuth; Arlot-Bonnemains, Yannick; Samuel, Didier; Gassama-Diagne, Ama

2016-12-06

Lumen formation during epithelial morphogenesis requires the creation of a luminal space at cell interfaces named apical membrane-initiation sites (AMISs). This is dependent upon integrated signaling from mechanical and biochemical cues, vesicle trafficking, cell division, and processes tightly coupled to ciliogenesis. Deciphering relationships between polarity determinants and lumen or cilia generation remains a fundamental issue. Here, we report that Src homology 2 domain-containing inositol 5-phosphatase 2 (SHIP2), a basolateral determinant of polarity, regulates RhoA-dependent actin contractility and cell division to form AMISs. SHIP2 regulates mitotic spindle alignment. SHIP2 is expressed in G1 phase, whereas Aurora A kinase is enriched in mitosis. SHIP2 binds Aurora A kinase and the scaffolding protein HEF1 and promotes their basolateral localization at the expense of their luminal expression connected with cilia resorption. Furthermore, SHIP2 expression increases cilia length. Thus, our findings offer new insight into the relationships among basolateral proteins, lumen generation, and ciliogenesis. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Modelling the fluid mechanics of cilia and flagella in reproduction and development.

PubMed

Montenegro-Johnson, Thomas D; Smith, Andrew A; Smith, David J; Loghin, Daniel; Blake, John R

2012-10-01

Cilia and flagella are actively bending slender organelles, performing functions such as motility, feeding and embryonic symmetry breaking. We review the mechanics of viscous-dominated microscale flow, including time-reversal symmetry, drag anisotropy of slender bodies, and wall effects. We focus on the fundamental force singularity, higher-order multipoles, and the method of images, providing physical insight and forming a basis for computational approaches. Two biological problems are then considered in more detail: 1) left-right symmetry breaking flow in the node, a microscopic structure in developing vertebrate embryos, and 2) motility of microswimmers through non-Newtonian fluids. Our model of the embryonic node reveals how particle transport associated with morphogenesis is modulated by the gradual emergence of cilium posterior tilt. Our model of swimming makes use of force distributions within a body-conforming finite-element framework, allowing the solution of nonlinear inertialess Carreau flow. We find that a three-sphere model swimmer and a model sperm are similarly affected by shear-thinning; in both cases swimming due to a prescribed beat is enhanced by shear-thinning, with optimal Deborah number around 0.8. The sperm exhibits an almost perfect linear relationship between velocity and the logarithm of the ratio of zero to infinite shear viscosity, with shear-thickening hindering cell progress.

Functional characterization of a novel 3D model of the epithelial-mesenchymal trophic unit.

PubMed

Bucchieri, Fabio; Pitruzzella, Alessandro; Fucarino, Alberto; Gammazza, Antonella Marino; Bavisotto, Celeste Caruso; Marcianò, Vito; Cajozzo, Massimo; Lo Iacono, Giorgio; Marchese, Roberto; Zummo, Giovanni; Holgate, Stephen T; Davies, Donna E

2017-03-01

Epithelial-mesenchymal communication plays a key role in tissue homeostasis and abnormal signaling contributes to chronic airways disease such as COPD. Most in vitro models are limited in complexity and poorly represent this epithelial-mesenchymal trophic unit. We postulated that cellular outgrowth from bronchial tissue would enable development of a mucosal structure that recapitulates better in vivo tissue architecture. Bronchial tissue was embedded in Matrigel and outgrowth cultures monitored using time-lapse microscopy, electrical resistance, light and electron microscopy. Cultures were challenged repetitively with cigarette smoke extract (CSE). The outgrowths formed as a multicellular sheet with motile cilia becoming evident as the Matrigel was remodeled to provide an air interface; cultures were viable for more than one year. Immunofluorescence and electron microscopy (EM) identified an upper layer of mucociliary epithelium and a lower layer of highly organized extracellular matrix (ECM) interspersed with fibroblastic cells separated by a basement membrane. EM analysis of the mucosal construct after repetitive exposure to CSE revealed epithelial damage, loss of cilia, and ECM remodeling, as occurs in vivo. We have developed a robust bronchial mucosal model. The structural changes observed following CSE exposure suggest the model should have utility for drug discovery and preclinical testing, especially those targeting airway remodeling.

A Numerical Model of Viscoelastic Layer Entrainment by Airflow in Cough

NASA Astrophysics Data System (ADS)

Mitran, Sorin M.

2008-07-01

Coughing is an alternative mode of ensuring mucus clearance in the lung when normal cilia induced flow breaks down. A numerical model of this process is presented with the following aspects. (1) A portion of the airway comprising the first three bronchus generations is modeled as radially reinforced elastic tubes. Elasticity equations are solved to predict airway deformation under effect of airway pressure. (2) The compressible, turbulent flow induced by rapid lung contraction is modeled by direct numerical simulation for Reynolds numbers in the range 5,000-10,000 and by Large Eddy Simulation for Reynolds numbers in the range 5,000-40,000. (3) A two-layer model of the airway surface liquid (ASL) covering the airway epithelial layer is used. The periciliary liquid (PCL) in direct contact with the epithelial layer is considered to be a Newtonian fluid. Forces modeling cilia beating can act upon this layer. The mucus layer between the PCL and the interior airflow is modeled as an Oldroyd-B fluid. The overall computation is a fluid-structure interaction simulation that tracks changes in ASL thickness and airway diameters that result from impulsive airflow boundary conditions imposed at bronchi ends. In particular, the amount of mucus that is evacuated from the system is computed as a function of cough intensity and mucus rheological properties.