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Sample records for polarised primary cilia

  1. Secreted frizzled-related protein disrupts PCP in eye lens fiber cells that have polarised primary cilia.

    PubMed

    Sugiyama, Yuki; Stump, Richard J W; Nguyen, Anke; Wen, Li; Chen, Yongjuan; Wang, Yanshu; Murdoch, Jennifer N; Lovicu, Frank J; McAvoy, John W

    2010-02-15

    Planar cell polarity (PCP) signaling polarises cells along tissue axes. Although pathways involved are becoming better understood, outstanding issues include; (i) existence/identity of cues that orchestrate global polarisation in tissues, and (ii) the generality of the link between polarisation of primary cilia and asymmetric localisation of PCP proteins. Mammalian lenses are mainly comprised of epithelial-derived fiber cells. Concentrically arranged fibers are precisely aligned as they elongate along the anterior-posterior axis and orientate towards lens poles where they meet fibers from other segments to form characteristic sutures. We show that lens exhibits PCP, with each fiber cell having an apically situated cilium and in most cases this is polarised towards the anterior pole. Frizzled and other PCP proteins are also asymmetrically localised along the equatorial-anterior axis. Mutations in core PCP genes Van Gogh-like 2 and Celsr1 perturb oriented fiber alignment and suture formation. Suppression of the PCP pathway by overexpressing Sfrp2 shows that whilst local groups of fibers are often similarly oriented, they lack global orientation; consequently when local groups of fibers with different orientations meet they form multiple, small, ectopic suture-like configurations. This indicates that this extracellular inhibitor disrupts a global polarising signal that utilises a PCP-mediated mechanism to coordinate the global alignment and orientation of fibers to lens poles. PMID:19968984

  2. Mechanical Properties of Primary Cilia

    NASA Astrophysics Data System (ADS)

    Battle, Christopher; Schmidt, Christoph F.

    2013-03-01

    Recent studies have shown that the primary cilium, long thought to be a vestigial cellular appendage with no function, is involved in a multitude of sensory functions. One example, interesting from both a biophysical and medical standpoint, is the primary cilium of kidney epithelial cells, which acts as a mechanosensitive flow sensor. Genetic defects in ciliary function can cause, e.g., polycystic kidney disease (PKD). The material properties of these non-motile, microtubule-based 9 +0 cilia, and the way they are anchored to the cell cytoskeleton, are important to know if one wants to understand the mechano-electrochemical response of these cells, which is mediated by their cilia. We have probed the mechanical properties, boundary conditions, and dynamics of the cilia of MDCK cells using optical traps and DIC/fluorescence microscopy. We found evidence for both elastic relaxation of the cilia themselves after bending and for compliance in the intracellular anchoring structures. Angular and positional fluctuations of the cilia reflect both thermal excitations and cellular driving forces.

  3. Primary cilia and forebrain development.

    PubMed

    Willaredt, Marc August; Tasouri, Evangelia; Tucker, Kerry L

    2013-01-01

    With a microtubule-based axoneme supporting its plasma membrane-ensheathed projection from the basal body of almost all cell types in the human body, and present in only one copy per cell, the primary cilium can be considered an organelle sui generis. Although it was first observed and recorded in histological studies from the late 19th century, the tiny structure was essentially forgotten for many decades. In the past ten years, however, scientists have turned their eyes once again upon primary cilia and realized that they are very important for the development of almost all organs in the mammalian body, especially those dependent upon the signaling from members Hedgehog family, such as Indian and Sonic hedgehog. In this review, we outline the roles that primary cilia play in forebrain development, not just in the crucial transduction of Sonic hedgehog signaling, but also new results showing that cilia are important for cell cycle progression in proliferating neural precursors. We will focus upon cerebral cortex development but will also discuss the importance of cilia for the embryonic hippocampus, olfactory bulb, and diencephalon. PMID:23085524

  4. Immunofluorescent staining of septins in primary cilia.

    PubMed

    Kim, M S; Froese, C D; Xie, H; Trimble, W S

    2016-01-01

    Primary cilia are cellular antennae that receive and transduce extracellular cues. These microtubule-rich structures are comprised of at least three distinct ciliary compartments: basal bodies, transition zone, and axoneme. Septins have been implicated in cilia function at the transition zone, but accumulating evidence suggests that they localize predominantly within the axoneme. Here, we describe three fixation conditions that preserve the substructure of primary cilia and demonstrate known ciliary proteins that localize to these distinct ciliary substructures. Finally, we show immunostaining and live microscopy methods to detect septins within the axoneme. PMID:27473914

  5. Primary Cilia in Pancreatic Development and Disease

    PubMed Central

    Lodh, Sukanya; O’Hare, Elizabeth A.; Zaghloul, Norann A.

    2014-01-01

    Primary cilia and their anchoring basal bodies are important regulators of a growing list of signaling pathways. Consequently, dysfunction in proteins associated with these structures results in perturbation of the development and function of a spectrum of tissue and cell types. Here, we review the role of cilia in mediating the development and function of the pancreas. We focus on ciliary regulation of major pathways involved in pancreatic development, including Shh, Wnt, TGF-β, Notch, and fibroblast growth factor. We also discuss pancreatic phenotypes associated with ciliary dysfunction, including pancreatic cysts and defects in glucose homeostasis, and explore the potential role of cilia in such defects. PMID:24864023

  6. Primary cilia utilize glycoprotein-dependent adhesion mechanisms to stabilize long-lasting cilia-cilia contacts

    PubMed Central

    2012-01-01

    Background The central tenet of cilia function is sensing and transmitting information. The capacity to directly contact extracellular surfaces would empower primary cilia to probe the environment for information about the nature and location of nearby surfaces. It has been well established that flagella and other motile cilia perform diverse cellular functions through adhesion. We hypothesized that mammalian primary cilia also interact with the extracellular environment through direct physical contact. Methods We identified cilia in rod photoreceptors and cholangiocytes in fixed mouse tissues and examined the structures that these cilia contact in vivo. We then utilized an MDCK cell culture model to characterize the nature of the contacts we observed. Results In retina and liver tissue, we observed that cilia from nearby cells touch one another. Using MDCK cells, we found compelling evidence that these contacts are stable adhesions that form bridges between two cells, or networks between many cells. We examined the nature and duration of the cilia-cilia contacts and discovered primary cilia movements that facilitate cilia-cilia encounters. Stable adhesions form as the area of contact expands from a single point to a stretch of tightly bound, adjacent cilia membranes. The cilia-cilia contacts persisted for hours and were resistant to several harsh treatments such as proteases and DTT. Unlike many other cell adhesion mechanisms, calcium was not required for the formation or maintenance of cilia adhesion. However, swainsonine, which blocks maturation of N-linked glycoproteins, reduced contact formation. We propose that cellular control of adhesion maintenance is active because cilia adhesion did not prevent cell division; rather, contacts dissolved during mitosis as cilia were resorbed. Conclusions The demonstration that mammalian primary cilia formed prolonged, direct, physical contacts supports a novel paradigm: that mammalian primary cilia detect features of the

  7. Detection of primary cilia in human glioblastoma

    PubMed Central

    Sarkisian, Matthew R.; Siebzehnrubl, Dorit; Hoang-Minh, Lan; Deleyrolle, Loic; Silver, Daniel J.; Siebzehnrubl, Florian A.; Guadiana, Sarah M.; Srivinasan, Gayathri; Semple-Rowland, Susan; Harrison, Jeffrey K.; Steindler, Dennis A.; Reynolds, Brent A.

    2015-01-01

    Glioblastoma (GBM) is the most common malignant adult brain tumor and carries a poor prognosis due to primary and acquired resistance. While many cellular features of GBM have been documented, it is unclear if cells within these tumors extend a primary cilium, an organelle whose associated signaling pathways may regulate proliferation, migration, and survival of neural precursor and tumor cells. Using immunohistochemical and electron microscopy (EM) techniques, we screened human GBM tumor biopsies and primary cell lines for cilia. Immunocytochemical staining of five primary GBM cell lines revealed that between 8 and 25 % of the cells in each line possessed gamma tubulin-positive basal bodies from which extended acetylated, alpha-tubulin-positive axonemes. EM analyses confirmed the presence of cilia at the cell surface and revealed that their axonemes contained organized networks of microtubules, a structural feature consistent with our detection of IFT88 and Arl13b, two trafficked cilia proteins, along the lengths of the axonemes. Notably, cilia were detected in each of 23 tumor biopsies (22 primary and 1 recurrent) examined. These cilia were distributed across the tumor landscape including regions proximal to the vasculature and within necrotic areas. Moreover, ciliated cells within these tumors co-stained with Ki67, a marker for actively dividing cells, and ZEB1, a transcription factor that is upregulated in GBM and linked to tumor initiation, invasion, and chemoresistance. Collectively, our data show that subpopulations of cells within human GBM tumors are ciliated. In view of mounting evidence supporting roles of primary cilia in tumor initiation and propagation, it is likely that further study of the effects of cilia on GBM tumor cell function will improve our understanding of GBM pathogenesis and may provide new directions for GBM treatment strategies. PMID:24510433

  8. The Roles of Primary cilia in Polycystic Kidney Disease

    PubMed Central

    Kathem, Sarmed H.; Mohieldin, Ashraf M.; Nauli, Surya M.

    2014-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is an inherited genetic disorder that results in progressive renal cyst formation with ultimate loss of renal function and other systemic disorders. These systemic disorders include abnormalities in cardiovascular, portal, pancreatic and gastrointestinal systems. ADPKD is considered to be among the ciliopathy diseases due to the association with abnormal primary cilia function. In order to understand the full course of primary cilia and its association with ADPKD, the structure, functions and role of primary cilia have been meticulously investigated. As a result, the focus on primary cilia has emerged to support the vital roles of primary cilia in ADPKD. The primary cilia have been shown to have not only a mechanosensory function but also a chemosensory function. Both structural and functional defects in primary cilia result in cystic kidney disease and vascular hypertension. Thus, the mechanosenory and chemosensory functions will be analyzed in regards to ADPKD. PMID:25599087

  9. Autophagy and primary cilia: dual interplay.

    PubMed

    Pampliega, Olatz; Cuervo, Ana Maria

    2016-04-01

    Primary cilia are microtubule-based organelles for sensing of the extracellular milieu and transducing this information into the cell through a variety of molecular signaling pathways. Functioning of the primary cilium has been recently connected to autophagy, a pathway for degradation of cellular components in lysosomes. Autophagy regulates the length of the cilia by removing proteins required for ciliogenesis, a phenomenon that is molecularly different if performed by basal autophagy or when autophagy is induced in response to various stressors. Here we review the current knowledge about the dual interaction between autophagy and ciliogenesis, and discuss the potential role that deregulated ciliary autophagy could have in pathologies with alterations in autophagy and ciliogenesis. PMID:26826446

  10. The emerging face of primary cilia

    PubMed Central

    Zaghloul, Norann A.; Brugmann, Samantha A.

    2011-01-01

    Primary cilia are microtubule-based organelles that serve as hubs for the transduction of various developmental signaling pathways including Hedgehog, Wnt, FGF and PDGF. Ciliary dysfunction contributes to a range of disorders, collectively known as the ciliopathies. Recently, interest has grown in these syndromes, particularly among craniofacial biologists, as many known and putative ciliopathies have severe craniofacial defects. Herein we discuss the current understanding of ciliary biology and craniofacial development in an attempt to gain insight into the molecular etiology for craniofacial ciliopathies, and uncover a characteristic ciliopathic craniofacial gestalt. PMID:21305689

  11. ADENYLATE CYCLASE REGULATES ELONGATION OF MAMMALIAN PRIMARY CILIA

    PubMed Central

    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J.; Rattner, Jerome B.; van der Hoorn, Frans A.

    2011-01-01

    The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3β by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1–2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway. PMID:19576885

  12. Adenylate cyclase regulates elongation of mammalian primary cilia

    SciTech Connect

    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J.; Rattner, Jerome B.; Hoorn, Frans A. van der

    2009-10-01

    The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3{beta} by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1-2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway.

  13. CLEM Methods for Studying Primary Cilia.

    PubMed

    Macaluso, Frank P; Perumal, Geoffrey S; Kolstrup, Johan; Satir, Peter

    2016-01-01

    CLEM (correlated light and electron microscope) imaging is a highly useful technique for examining primary cilia. With CLEM, it is possible to determine the distribution of tagged proteins along the ciliary membrane and axoneme with high precision. Scanning electron microscopy (SEM) permits measurement of ciliary length and orientation in relation to nearby cellular structures in a 3D image; in optimal cases, this can be combined with superresolution microscopy of selected ciliary components as they enter or leave the cilium. This chapter discusses CLEM methods. In the method described in detail, samples are completely processed for sequential fluorescence and SEM observation. This method is ideal for robust antibody localization and minimizes image manipulation in correlating the fluorescent and SEM images. Alternative methods prepare samples for fluorescence imaging followed by processing for SEM then observation in the SEM. This method is ideal for optimal fluorescence imaging, particularly live cell imaging. PMID:27514923

  14. Electrical Signaling in Motile and Primary Cilia

    PubMed Central

    Kleene, Steven J.; Van Houten, Judith L.

    2014-01-01

    Cilia are highly conserved for their structure and also for their sensory functions. They serve as antennae for extracellular information. Whether the cilia are motile or not, they respond to environmental mechanical and chemical stimuli and send signals to the cell body. The information from extracellular stimuli is commonly converted to electrical signals through the repertoire of ion-conducting channels in the ciliary membrane, which results in changes in concentrations of ions, especially calcium ions, in the cilia. These changes, in turn, affect motility and the ability of the signaling pathways in the cilia and cell body to carry on the signal transduction. We review here the activities of ion channels in cilia in animals from protists to vertebrates. PMID:25892740

  15. Visualization of live primary cilia dynamics using fluorescence microscopy

    PubMed Central

    Ott, Carolyn; Lippincott-Schwartz, Jennifer

    2013-01-01

    Here we describe methods that are useful for exploring the formation and functions of primary cilia in living cells. First we describe multiple protocols for visualizing solitary cilia that extend away from the cell body. Primary cilia collect, synthesize, and transmit information about the extracellular space into the cell body to promote critical cellular responses. Problems with cilia formation or function can lead to dramatic changes in cell physiology. These methods can be used to assess cilia formation and length, the location of the cilium relative to other cellular structures, and localization of specific proteins to the cilium. The second protocol describes how to quantify movement of fluorescent molecules within the cilium. The microtubules that form the structural scaffold of the cilium are also critical avenues for kinesin and dynein-mediated movement of proteins within the cilium. Assessing intraflagellar dynamics can provide insight into mechanisms of ciliary-mediated signal perception and transmission. PMID:23208547

  16. Primary cilia are not calcium-responsive mechanosensors.

    PubMed

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

    2016-03-31

    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 cilium. This Ca(2+)-responsive mechanosensor 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 cancers. Here we report the complete lack of mechanically induced calcium increases in primary cilia, in tissues upon which this hypothesis has been based. We developed a transgenic mouse, Arl13b-mCherry-GECO1.2, expressing a ratiometric genetically encoded calcium indicator 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 Ca(2+) 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 signalling. PMID:27007841

  17. Primary cilia and autophagic dysfunction in Huntington's disease.

    PubMed

    Kaliszewski, M; Knott, A B; Bossy-Wetzel, E

    2015-09-01

    Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by a single-gene mutation: a CAG expansion in the huntingtin (HTT) gene that results in production of a mutated protein, mutant HTT, with a polyglutamine tail (polyQ-HTT). Although the molecular pathways of polyQ-HTT toxicity are not fully understood, because protein misfolding and aggregation are central features of HD, it has long been suspected that cellular housekeeping processes such as autophagy might be important to disease pathology. Indeed, multiple lines of research have identified abnormal autophagy in HD, characterized generally by increased autophagic induction and inefficient clearance of substrates. To date, the origin of autophagic dysfunction in HD remains unclear and the search for actors involved continues. To that end, recent studies have suggested a bidirectional relationship between autophagy and primary cilia, signaling organelles of most mammalian cells. Interestingly, primary cilia structure is defective in HD, suggesting a potential link between autophagic dysfunction, primary cilia and HD pathogenesis. In addition, because polyQ-HTT also accumulates in primary cilia, the possibility exists that primary cilia might play additional roles in HD: perhaps by disrupting signaling pathways or acting as a reservoir for secretion and propagation of toxic, misfolded polyQ-HTT fragments. Here, we review recent research suggesting potential links between autophagy, primary cilia and HD and speculate on possible pathogenic mechanisms and future directions for the field. PMID:26160070

  18. Analysis of primary cilia in the developing mouse brain.

    PubMed

    Paridaen, Judith T M L; Huttner, Wieland B; Wilsch-Bräuninger, Michaela

    2015-01-01

    Stem and progenitor cells in the developing mammalian brain are highly polarized cells that carry a primary cilium protruding into the brain ventricles. Here, cilia detect signals present in the cerebrospinal fluid that fills the ventricles. Recently, striking observations have been made regarding the dynamics of primary cilia in mitosis and cilium reformation after cell division. In neural progenitors, primary cilia are not completely disassembled during cell division, and some ciliary membrane remnant can be inherited by one daughter cell that tends to maintain a progenitor fate. Furthermore, newborn differentiating cells grow a primary cilium on their basolateral plasma membrane, in spite of them possessing apical membrane and adherens junctions, and thus change the environment to which the primary cilium is exposed. These phenomena are proposed to be involved in cell fate determination and delamination of daughter cells in conjunction with the production of neurons. Here, we describe several methods that can be used to study the structure, localization, and dynamics of primary cilia in the developing mouse brain; these include time-lapse imaging of live mouse embryonic brain tissues, and analysis of primary cilia structure and localization using correlative light- and electron- and serial-block-face scanning electron microscopy. PMID:25837388

  19. Primary cilia and Gli3 activity regulate cerebral cortical size

    PubMed Central

    Wilson, Sandra L.; Wilson, John P.; Wang, Chengbing; Wang, Baolin; McConnell, Susan K.

    2012-01-01

    During neural development, patterning, neurogenesis and overall growth are highly regulated and coordinated between different brain regions. Here, we show that primary cilia and the regulation of Gli activity, are necessary for the normal expansion of the cerebral cortex. We show that loss of Kif3a, an important functional component of primary cilia, leads to the degeneration of primary cilia, marked overgrowth of the cortex, and altered cell cycle kinetics within cortical progenitors. The G1 phase of the cell cycle is shortened through a mechanism likely involving reduced Gli3 activity and a resulting increase in expression of cyclin D1 and Fgf15. The defects in Gli3 activity alone are sufficient to accelerate cell cycle kinetics and cause the molecular changes seen in brains that lack cilia. Finally, we show that levels of full-length and repressor Gli3 proteins are tightly regulated during normal development and correlate with changes in expression of two known Shh-target genes, CyclinD1 and Fgf15, and with the normal lengthening of the cell cycle during corticogenesis. These data suggest that Gli3 activity is regulated through the primary cilium to control cell cycle length in the cortex and thus determine cortical size. PMID:21976438

  20. Ins and outs of GPCR signaling in primary cilia.

    PubMed

    Schou, Kenneth Bødtker; Pedersen, Lotte Bang; Christensen, Søren Tvorup

    2015-09-01

    Primary cilia are specialized microtubule-based signaling organelles that convey extracellular signals into a cellular response in most vertebrate cell types. The physiological significance of primary cilia is underscored by the fact that defects in assembly or function of these organelles lead to a range of severe diseases and developmental disorders. In most cell types of the human body, signaling by primary cilia involves different G protein-coupled receptors (GPCRs), which transmit specific signals to the cell through G proteins to regulate diverse cellular and physiological events. Here, we provide an overview of GPCR signaling in primary cilia, with main focus on the rhodopsin-like (class A) and the smoothened/frizzled (class F) GPCRs. We describe how such receptors dynamically traffic into and out of the ciliary compartment and how they interact with other classes of ciliary GPCRs, such as class B receptors, to control ciliary function and various physiological and behavioral processes. Finally, we discuss future avenues for developing GPCR-targeted drug strategies for the treatment of ciliopathies. PMID:26297609

  1. Intracellular and extracellular forces drive primary cilia movement

    PubMed Central

    Battle, Christopher; Ott, Carolyn M.; Burnette, Dylan T.; Lippincott-Schwartz, Jennifer; Schmidt, Christoph F.

    2015-01-01

    Primary cilia are ubiquitous, microtubule-based organelles that play diverse roles in sensory transduction in many eukaryotic cells. They interrogate the cellular environment through chemosensing, osmosensing, and mechanosensing using receptors and ion channels in the ciliary membrane. Little is known about the mechanical and structural properties of the cilium and how these properties contribute to ciliary perception. We probed the mechanical responses of primary cilia from kidney epithelial cells [Madin–Darby canine kidney-II (MDCK-II)], which sense fluid flow in renal ducts. We found that, on manipulation with an optical trap, cilia deflect by bending along their length and pivoting around an effective hinge located below the basal body. The calculated bending rigidity indicates weak microtubule doublet coupling. Primary cilia of MDCK cells lack interdoublet dynein motors. Nevertheless, we found that the organelles display active motility. 3D tracking showed correlated fluctuations of the cilium and basal body. These angular movements seemed random but were dependent on ATP and cytoplasmic myosin-II in the cell cortex. We conclude that force generation by the actin cytoskeleton surrounding the basal body results in active ciliary movement. We speculate that actin-driven ciliary movement might tune and calibrate ciliary sensory functions. PMID:25605896

  2. Intracellular and extracellular forces drive primary cilia movement.

    PubMed

    Battle, Christopher; Ott, Carolyn M; Burnette, Dylan T; Lippincott-Schwartz, Jennifer; Schmidt, Christoph F

    2015-02-01

    Primary cilia are ubiquitous, microtubule-based organelles that play diverse roles in sensory transduction in many eukaryotic cells. They interrogate the cellular environment through chemosensing, osmosensing, and mechanosensing using receptors and ion channels in the ciliary membrane. Little is known about the mechanical and structural properties of the cilium and how these properties contribute to ciliary perception. We probed the mechanical responses of primary cilia from kidney epithelial cells [Madin-Darby canine kidney-II (MDCK-II)], which sense fluid flow in renal ducts. We found that, on manipulation with an optical trap, cilia deflect by bending along their length and pivoting around an effective hinge located below the basal body. The calculated bending rigidity indicates weak microtubule doublet coupling. Primary cilia of MDCK cells lack interdoublet dynein motors. Nevertheless, we found that the organelles display active motility. 3D tracking showed correlated fluctuations of the cilium and basal body. These angular movements seemed random but were dependent on ATP and cytoplasmic myosin-II in the cell cortex. We conclude that force generation by the actin cytoskeleton surrounding the basal body results in active ciliary movement. We speculate that actin-driven ciliary movement might tune and calibrate ciliary sensory functions. PMID:25605896

  3. Primary cilia and kidney injury: current research status and future perspectives

    PubMed Central

    Wang, Shixuan

    2013-01-01

    Cilia, membrane-enclosed organelles protruding from the apical side of cells, can be divided into two classes: motile and primary cilia. During the past decades, motile cilia have been intensively studied. However, it was not until the 1990s that people began to realize the importance of primary cilia as cellular-specific sensors, particularly in kidney tubular epithelial cells. Furthermore, accumulating evidence indicates that primary cilia may be involved in the regulation of cell proliferation, differentiation, apoptosis, and planar cell polarity. Many signaling pathways, such as Wnt, Notch, Hedgehog, and mammalian target of rapamycin, have been located to the primary cilia. Thus primary cilia have been regarded as a hub that integrates signals from the extracellular environment. More importantly, dysfunction of this organelle may contribute to the pathogenesis of a large spectrum of human genetic diseases, named ciliopathies. The significance of primary cilia in acquired human diseases such as hypertension and diabetes has gradually drawn attention. Interestingly, recent reports disclosed that cilia length varies during kidney injury, and shortening of cilia enhances the sensitivity of epithelial cells to injury cues. This review briefly summarizes the current status of cilia research and explores the potential mechanisms of cilia-length changes during kidney injury as well as provides some thoughts to allure more insightful ideas and promotes the further study of primary cilia in the context of kidney injury. PMID:23904226

  4. Primary Cilia Regulate Branching Morphogenesis During Mammary Gland Development

    PubMed Central

    McDermott, Kimberly M.; Liu, Bob Y.; Tlsty, Thea D.; Pazour, Gregory J.

    2010-01-01

    Summary During mammary gland development an epithelial bud undergoes branching morphogenesis to expand into a continuous tree-like network of branched ducts [1]. The process involves multiple cell types that are coordinated by hormones and growth factors coupled with signaling events including Wnt and Hedgehog [2-5]. Primary cilia play key roles in the development of many organs by coordinating extracellular signaling (Wnt, Hedgehog) with cellular physiology [6-8]. During mammary development, we find cilia on luminal epithelial, myoepithelial and stromal cells during early branching morphogenesis when epithelial ducts extend into the fat pad and undergo branching morphogenesis. When branching is complete, cilia disappear from luminal epithelial cells but remain on myoepithelial and stromal cells. Ciliary dysfunction caused by intraflagellar transport (IFT) defects results in branching defects. These include decreased ductal extension and decreased secondary and tertiary branching along with reduced lobular-alveolar development during pregnancy and lactation. We find increased canonical Wnt and decreased Hedgehog signaling in the mutant glands, which is consistent with the role of cilia in regulating these pathways [6-11]. In mammary gland and other organs, increased canonical Wnt [12-14] and decreased Hedgehog [15, 16] signaling decreases branching morphogenesis suggesting that Wnt and Hedgehog signaling connect ciliary dysfunction to branching defects. PMID:20381354

  5. Characterization of cancer stem cells and primary cilia in medulloblastoma.

    PubMed

    Gate, David; Danielpour, Moise; Bannykh, Serguei; Town, Terrence

    2015-01-01

    Medulloblastoma, a tumor of the cerebellum, is the most common pediatric central nervous system malignancy. These tumors are etiologically linked to mutations in the Sonic hedgehog (Shh) pathway, which signals through the primary, non-motile cilium. The growth of these aggressive tumors relies on self-renewal of tumor-propagating cells known as cancer stem cells (CSCs). Previous reports have implicated CD133-expressing cells as CSCs in brain tumors, while those expressing CD15 have been shown to propagate medulloblastoma. Here, we demonstrate that CD133+ and CD15+ cells are distinct medulloblastoma populations. CD15+ cells comprise approximately 0.5-1% of total human medulloblastoma cells, display CSC properties in culture and are detected in the Smoothened A1 transgenic mouse model of medulloblastoma. Additionally, we report on a medulloblastoma patient with enriched CD15+ cells in recurrent vs primary medulloblastoma. We also demonstrate that human medulloblastoma cells critically rely on establishment of primary cilia to drive Shh-mediated cell division. Primary cilia are found in external granule cells of human fetal cerebellum and in 12/14 medulloblastoma samples. Yet, CD15+ medulloblastoma cells lack primary cilia, suggesting that this CSC population signals independently of Shh. These results are important when considering the effects of current and prospective treatment modalities on medulloblastoma CSC populations. PMID:25921740

  6. Oscillatory Fluid Flow Influences Primary Cilia and Microtubule Mechanics

    PubMed Central

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

    2014-01-01

    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. PMID:25044764

  7. Primary cilia in energy balance signaling and metabolic disorder

    PubMed Central

    Lee, Hankyu; Song, Jieun; Jung, Joo Hyun; Ko, Hyuk Wan

    2015-01-01

    Energy homeostasis in our body system is maintained by balancing the intake and expenditure of energy. Excessive accumulation of fat by disrupting the balance system causes overweight and obesity, which are increasingly becoming global health concerns. Understanding the pathogenesis of obesity focused on studying the genes related to familial types of obesity. Recently, a rare human genetic disorder, ciliopathy, links the role for genes regulating structure and function of a cellular organelle, the primary cilium, to metabolic disorder, obesity and type II diabetes. Primary cilia are microtubule based hair-like membranous structures, lacking motility and functions such as sensing the environmental cues, and transducing extracellular signals within the cells. Interestingly, the subclass of ciliopathies, such as Bardet-Biedle and Alström syndrome, manifest obesity and type II diabetes in human and mouse model systems. Moreover, studies on genetic mouse model system indicate that more ciliary genes affect energy homeostasis through multiple regulatory steps such as central and peripheral actions of leptin and insulin. In this review, we discuss the latest findings in primary cilia and metabolic disorders, and propose the possible interaction between primary cilia and the leptin and insulin signal pathways which might enhance our understanding of the unambiguous link of a cell’s antenna to obesity and type II diabetes. [BMB Reports 2015; 48(12): 647-654] PMID:26538252

  8. Function and regulation of primary cilia and intraflagellar transport proteins in the skeleton

    PubMed Central

    Yuan, Xue; Serra, Rosa A.; Yang, Shuying

    2014-01-01

    Primary cilia are microtubule-based organelles that project from the cell surface to enable transduction of various developmental signaling pathways. The process of intraflagellar transport (IFT) is crucial for the building and maintenance of primary cilia. Ciliary dysfunction has been found in a range of disorders called ciliopathies, some of which display severe skeletal dysplasias. In recent years, interest has grown in uncovering the function of primary cilia/IFT proteins in bone development, mechanotransduction, and cellular regulation. We summarize recent advances in understanding the function of cilia and IFT proteins in the regulation of cell differentiation in osteoblasts, osteocytes, chondrocytes, and mesenchymal stem cells (MSCs). We also discuss the mechanosensory function of cilia and IFT proteins in bone cells, cilia orientation, and other functions of cilia in chondrocytes. PMID:24961486

  9. Molecular complexes that direct rhodopsin transport to primary cilia

    PubMed Central

    Wang, Jing; Deretic, Dusanka

    2013-01-01

    Rhodopsin is a key molecular constituent of photoreceptor cells, yet understanding of how it regulates photoreceptor membrane trafficking and biogenesis of light-sensing organelles, the rod outer segments (ROS) is only beginning to emerge. Recently identified sequence of well-orchestrated molecular interactions of rhodopsin with the functional networks of Arf and Rab GTPases at multiple stages of intracellular targeting fits well into the complex framework of the biogenesis and maintenance of primary cilia, of which the ROS is one example. This review will discuss the latest progress in dissecting the molecular complexes that coordinate rhodopsin incorporation into ciliary-targeted carriers with the recruitment and activation of membrane tethering complexes and regulators of fusion with the periciliary plasma membrane. In addition to revealing the fundamental principals of ciliary membrane renewal, recent advances also provide molecular insight into the ways by which disruptions of the exquisitely orchestrated interactions lead to cilia dysfunction and result in human retinal dystrophies and syndromic diseases that affect multiple organs, including the eyes. PMID:24135424

  10. Unilateral nephrectomy elongates primary cilia in the remaining kidney via reactive oxygen species

    PubMed Central

    Han, Sang Jun; Jang, Hee-Seong; Kim, Jee In; Lipschutz, Joshua H.; Park, Kwon Moo

    2016-01-01

    The length of primary cilia is associated with normal cell and organ function. In the kidney, the change of functional cilia length/mass is associated with various diseases such as ischemia/reperfusion injury, polycystic kidney disease, and congenital solitary kidney. Here, we investigate whether renal mass reduction affects primary cilia length and function. To induce renal mass reduction, mice were subjected to unilateral nephrectomy (UNx). UNx increased kidney weight and superoxide formation in the remaining kidney. Primary cilia were elongated in proximal tubule cells, collecting duct cells and parietal cells of the remaining kidney. Mn(III) Tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP), an antioxidant, reduced superoxide formation in UNx-mice and prevented the elongation of primary cilia. UNx increased the expression of phosphorylated ERK, p21, and exocyst complex members Sec8 and Sec10, in the remaining kidney, and these increases were prevented by MnTMPyP. In MDCK, a kidney tubular epithelial cell line, cells, low concentrations of H2O2 treatment elongated primary cilia. This H2O2-induced elongation of primary cilia was also prevented by MnTMPyP treatment. Taken together, these data demonstrate that kidney compensation, induced by a reduction of renal mass, results in primary cilia elongation, and this elongation is associated with an increased production of reactive oxygen species (ROS). PMID:26923764

  11. Developmental changes in primary cilia in the mouse tooth germ and oral cavity.

    PubMed

    Hisamoto, Meri; Goto, Marie; Muto, Mami; Nio-Kobayashi, Junko; Iwanaga, Toshihiko; Yokoyama, Atsuro

    2016-01-01

    The primary cilium, a sensory apparatus, functions as both a chemical and mechanical sensor to receive environmental stimuli. The present study focused on the primary cilia in the epithelialmesenchymal interaction during tooth development. We examined the localization and direction of projection of primary cilia in the tooth germ and oral cavity of mice by immunohistochemical observation. Adenylyl cyclase 3 (ACIII)-immunolabeled cilia were visible in the inner/outer enamel epithelium of molars at the fetal stage and then conspicuously developed in the odontoblast layer postnatally. The primary cilia in ameloblasts and odontoblasts-shown by the double staining of acetylated tubulin and γ-tubulin-were regularly arranged from postnatal Day12, projecting apart from each other. The periodontal ligament possessed ACIII-positive cilia, which gathered on both sides of the dentin/cement and alveolar bone in postnatal days. In the oral cavity, numerous long primary cilia immunoreactive for ACIII were condensed at subepithelial stromal cells in the oral processes in fetuses, while postnatally a small number of short cilia were dispersed throughout the stroma of the oral cavity. These findings suggest that the primary cilia showing stage- and regionspecific morphology are involved in the epithelial-mesenchymal interaction during tooth development via mechano- and/or chemoreception for growth factors. PMID:27356608

  12. Primary cilia are highly oriented with respect to collagen direction and long axis of extensor tendon.

    PubMed

    Donnelly, Eve; Ascenzi, Maria-Grazia; Farnum, Cornelia

    2010-01-01

    Skeletal tissues adapt to their mechanical environments by modulating gene expression, cell metabolism, and extracellular matrix (ECM) architecture; however, the mechanosensory mechanisms for these processes are incompletely understood. Primary cilia have emerged as critical components of the cellular mechanosensory apparatus and have been hypothesized to participate in establishment of cellular and ECM orientation, but their function in skeletal tissues is just beginning to be examined. Here we focused on tendon, a tissue with an oriented matrix that is ideal for analysis of spatial relationships between primary cilia and the ECM. The objective of this study was to characterize the incidence and orientation of tenocyte primary cilia in their native ECM. Primary cilia, nuclei, and collagen were analyzed three-dimensionally in immunofluorescently labeled rat extensor tendon using multiphoton microscopy and semiautomated morphometry. Primary cilia were observed in 64% of tenocytes. The cilia were highly oriented with respect to the ECM: cilia were aligned parallel to the collagen fibers and the long axis of the tendon. This study represents the first quantification of the in situ incidence and orientation of primary cilia in tendon. PMID:19603516

  13. Surface topography regulates wnt signaling through control of primary cilia structure in mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    McMurray, R. J.; Wann, A. K. T.; Thompson, C. L.; Connelly, J. T.; Knight, M. M.

    2013-12-01

    The primary cilium regulates cellular signalling including influencing wnt sensitivity by sequestering β-catenin within the ciliary compartment. Topographic regulation of intracellular actin-myosin tension can control stem cell fate of which wnt is an important mediator. We hypothesized that topography influences mesenchymal stem cell (MSC) wnt signaling through the regulation of primary cilia structure and function. MSCs cultured on grooves expressed elongated primary cilia, through reduced actin organization. siRNA inhibition of anterograde intraflagellar transport (IFT88) reduced cilia length and increased active nuclear β-catenin. Conversely, increased primary cilia assembly in MSCs cultured on the grooves was associated with decreased levels of nuclear active β-catenin, axin-2 induction and proliferation, in response to wnt3a. This negative regulation, on grooved topography, was reversed by siRNA to IFT88. This indicates that subtle regulation of IFT and associated cilia structure, tunes the wnt response controlling stem cell differentiation.

  14. Comparative study of the primary cilia in thyrocytes of adult mammals.

    PubMed

    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

    2015-10-01

    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

  15. Direct recording and molecular identification of the calcium channel of primary cilia

    NASA Astrophysics Data System (ADS)

    Decaen, Paul G.; Delling, Markus; Vien, Thuy N.; Clapham, David E.

    2013-12-01

    A primary cilium is a solitary, slender, non-motile protuberance of structured microtubules (9+0) enclosed by plasma membrane. Housing components of the cell division apparatus between cell divisions, primary cilia also serve as specialized compartments for calcium signalling and hedgehog signalling pathways. Specialized sensory cilia such as retinal photoreceptors and olfactory cilia use diverse ion channels. An ion current has been measured from primary cilia of kidney cells, but the responsible genes have not been identified. The polycystin proteins (PC and PKD), identified in linkage studies of polycystic kidney disease, are candidate channels divided into two structural classes: 11-transmembrane proteins (PKD1, PKD1L1 and PKD1L2) remarkable for a large extracellular amino terminus of putative cell adhesion domains and a G-protein-coupled receptor proteolytic site, and the 6-transmembrane channel proteins (PKD2, PKD2L1 and PKD2L2; TRPPs). Evidence indicates that the PKD1 proteins associate with the PKD2 proteins via coiled-coil domains. Here we use a transgenic mouse in which only cilia express a fluorophore and use it to record directly from primary cilia, and demonstrate that PKD1L1 and PKD2L1 form ion channels at high densities in several cell types. In conjunction with an accompanying manuscript, we show that the PKD1L1-PKD2L1 heteromeric channel establishes the cilia as a unique calcium compartment within cells that modulates established hedgehog pathways.

  16. Tubulin glycylases are required for primary cilia, control of cell proliferation and tumor development in colon

    PubMed Central

    Rocha, Cecilia; Papon, Laura; Cacheux, Wulfran; Marques Sousa, Patricia; Lascano, Valeria; Tort, Olivia; Giordano, Tiziana; Vacher, Sophie; Lemmers, Benedicte; Mariani, Pascale; Meseure, Didier; Medema, Jan Paul; Bièche, Ivan; Hahne, Michael; Janke, Carsten

    2014-01-01

    TTLL3 and TTLL8 are tubulin glycine ligases catalyzing posttranslational glycylation of microtubules. We show here for the first time that these enzymes are required for robust formation of primary cilia. We further discover the existence of primary cilia in colon and demonstrate that TTLL3 is the only glycylase in this organ. As a consequence, colon epithelium shows a reduced number of primary cilia accompanied by an increased rate of cell division in TTLL3-knockout mice. Strikingly, higher proliferation is compensated by faster tissue turnover in normal colon. In a mouse model for tumorigenesis, lack of TTLL3 strongly promotes tumor development. We further demonstrate that decreased levels of TTLL3 expression are linked to the development of human colorectal carcinomas. Thus, we have uncovered a novel role for tubulin glycylation in primary cilia maintenance, which controls cell proliferation of colon epithelial cells and plays an essential role in colon cancer development. PMID:25180231

  17. Tubulin glycylases are required for primary cilia, control of cell proliferation and tumor development in colon.

    PubMed

    Rocha, Cecilia; Papon, Laura; Cacheux, Wulfran; Marques Sousa, Patricia; Lascano, Valeria; Tort, Olivia; Giordano, Tiziana; Vacher, Sophie; Lemmers, Benedicte; Mariani, Pascale; Meseure, Didier; Medema, Jan Paul; Bièche, Ivan; Hahne, Michael; Janke, Carsten

    2014-10-01

    TTLL3 and TTLL8 are tubulin glycine ligases catalyzing posttranslational glycylation of microtubules. We show here for the first time that these enzymes are required for robust formation of primary cilia. We further discover the existence of primary cilia in colon and demonstrate that TTLL3 is the only glycylase in this organ. As a consequence, colon epithelium shows a reduced number of primary cilia accompanied by an increased rate of cell division in TTLL3-knockout mice. Strikingly, higher proliferation is compensated by faster tissue turnover in normal colon. In a mouse model for tumorigenesis, lack of TTLL3 strongly promotes tumor development. We further demonstrate that decreased levels of TTLL3 expression are linked to the development of human colorectal carcinomas. Thus, we have uncovered a novel role for tubulin glycylation in primary cilia maintenance, which controls cell proliferation of colon epithelial cells and plays an essential role in colon cancer development. PMID:25180231

  18. Primary Cilia Integrate Hedgehog and Wnt Signaling during Tooth Development

    PubMed Central

    Liu, B.; Chen, S.; Cheng, D.; Jing, W.; Helms, J.A.

    2014-01-01

    Many ciliopathies have clinical features that include tooth malformations but how these defects come about is not clear. Here we show that genetic deletion of the motor protein Kif3a in dental mesenchyme results in an arrest in odontogenesis. Incisors are completely missing, and molars are enlarged in Wnt1Cre+Kif3afl/fl embryos. Although amelogenesis and dentinogenesis initiate in the molar tooth bud, both processes terminate prematurely. We demonstrate that loss of Kif3a in dental mesenchyme results in loss of Hedgehog signaling and gain of Wnt signaling in this same tissue. The defective dental mesenchyme then aberrantly signals to the dental epithelia, which prompts an up-regulation in the Hedgehog and Wnt responses in the epithelia and leads to multiple attempts at invagination and an expanded enamel organ. Thus, the primary cilium integrates Hedgehog and Wnt signaling between dental epithelia and mesenchyme, and this cilia-dependent integration is required for proper tooth development. PMID:24659776

  19. Primary Cilia on Horizontal Basal Cells Regulate Regeneration of the Olfactory Epithelium

    PubMed Central

    Joiner, Ariell M.; Green, Warren W.; McIntyre, Jeremy C.; Allen, Benjamin L.; Schwob, James E.

    2015-01-01

    The olfactory epithelium (OE) is one of the few tissues to undergo constitutive neurogenesis throughout the mammalian lifespan. It is composed of multiple cell types including olfactory sensory neurons (OSNs) that are readily replaced by two populations of basal stem cells, frequently dividing globose basal cells and quiescent horizontal basal cells (HBCs). However, the precise mechanisms by which these cells mediate OE regeneration are unclear. Here, we show for the first time that the HBC subpopulation of basal stem cells uniquely possesses primary cilia that are aligned in an apical orientation in direct apposition to sustentacular cell end feet. The positioning of these cilia suggests that they function in the detection of growth signals and/or differentiation cues. To test this idea, we generated an inducible, cell type-specific Ift88 knock-out mouse line (K5rtTA;tetOCre;Ift88fl/fl) to disrupt cilia formation and maintenance specifically in HBCs. Surprisingly, the loss of HBC cilia did not affect the maintenance of the adult OE but dramatically impaired the regeneration of OSNs following lesion. Furthermore, the loss of cilia during development resulted in a region-specific decrease in neurogenesis, implicating HBCs in the establishment of the OE. Together, these results suggest a novel role for primary cilia in HBC activation, proliferation, and differentiation. SIGNIFICANCE STATEMENT We show for the first time the presence of primary cilia on a quiescent population of basal stem cells, the horizontal basal cells (HBCs), in the olfactory epithelium (OE). Importantly, our data demonstrate that cilia on HBCs are necessary for regeneration of the OE following injury. Moreover, the disruption of HBC cilia alters neurogenesis during the development of the OE, providing evidence that HBCs participate in the establishment of this tissue. These data suggest that the mechanisms of penetrance for ciliopathies in the OE extend beyond that of defects in olfactory sensory

  20. Modulation of primary cilia length by melanin-concentrating hormone receptor 1.

    PubMed

    Hamamoto, Akie; Yamato, Shogo; Katoh, Yohei; Nakayama, Kazuhisa; Yoshimura, Kentaro; Takeda, Sen; Kobayashi, Yuki; Saito, Yumiko

    2016-06-01

    Melanin-concentrating hormone (MCH) receptor 1 (MCHR1) is a class A G-protein-coupled receptor (GPCR). The MCH-MCHR1 system has been implicated in the regulation of feeding, emotional processing, and sleep in rodents. Recent work revealed that MCHR1 is selectively expressed in neuronal primary cilia of the central nervous system. Cilia have various chemosensory functions in many types of cell, and ciliary dysfunction is associated with ciliopathies such as polycystic kidney disease and obesity. Although dynamic modulation of neuronal cilia length is observed in obese mice, the functional interaction of neuronal ciliary GPCR and its endogenous ligand has not yet been elucidated. We report here that MCH treatment significantly reduced cilia length in hTERT-RPE1 cells (hRPE1 cells) transfected with MCHR1. Quantitative analyses indicated that MCH-induced cilia shortening progressed in a dose-dependent manner with an EC50 lower than 1nM when cells were treated for 6h. Although the assembly and disassembly of primary cilia are tightly coupled to the cell cycle, cell cycle reentry was not a determinant of MCH-induced cilia shortening. We confirmed that MCH elicited receptor internalization, Ca(2+) mobilization, ERK and Akt phosphorylation, and inhibition of cyclic AMP accumulation in MCHR1-expressing hRPE1 cells. Among these diverse pathways, we revealed that Gi/o-dependent Akt phosphorylation was an important component in the initial stage of MCH-induced cilia length shortening. Furthermore, induction of fewer cilia by Kif3A siRNA treatment significantly decreased the MCH-mediated phosphorylation of Akt, indicating the functional importance of the MCHR1-Akt pathway in primary cilia. Taken together, the present data suggest that the MCH-MCHR1 axis may modulate the sensitivity of cells to external environments by controlling the cilia length. Therefore, further characterization of MCHR1 as a ciliary GPCR will provide a potential molecular mechanism to link cilia length

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

    SciTech Connect

    Miyoshi, Ko; Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato

    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 with 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.

  2. Hippocampal and Cortical Primary Cilia Are Required for Aversive Memory in Mice

    PubMed Central

    Yazdi, S. M. Zaki R.; McNair, Andrew D.; Kippe, Jordyn M.; Croyle, Mandy J.; Kraft, Timothy W.; Yoder, Bradley K.

    2014-01-01

    It has been known for decades that neurons throughout the brain possess solitary, immotile, microtubule based appendages called primary cilia. Only recently have studies tried to address the functions of these cilia and our current understanding remains poor. To determine if neuronal cilia have a role in behavior we specifically disrupted ciliogenesis in the cortex and hippocampus of mice through conditional deletion of the Intraflagellar Transport 88 (Ift88) gene. The effects on learning and memory were analyzed using both Morris Water Maze and fear conditioning paradigms. In comparison to wild type controls, cilia mutants displayed deficits in aversive learning and memory and novel object recognition. Furthermore, hippocampal neurons from mutants displayed an altered paired-pulse response, suggesting that loss of IFT88 can alter synaptic properties. A variety of other behavioral tests showed no significant differences between conditional cilia mutants and controls. This type of conditional allele approach could be used to distinguish which behavioral features of ciliopathies arise due to defects in neural development and which result from altered cell physiology. Ultimately, this could lead to an improved understanding of the basis for the cognitive deficits associated with human cilia disorders such as Bardet-Biedl syndrome, and possibly more common ailments including depression and schizophrenia. PMID:25184295

  3. RC/BTB2 is Essential for Formation of Primary Cilia in Mammalian Cells

    PubMed Central

    Zhang, Ling; Li, Wei; Ni, Jin; Wu, Jinghua; Liu, Junping; Zhang, Zhengang; Zhang, Yong; Li, Hongfei; Shi, Yuqin; Teves, Maria E; Song, Shizheng; Strauss, Jerome F.; Zhang, Zhibing

    2016-01-01

    RC/BTB2 is a binding partner of sperm associated antigen 16S (SPAG16S), which is regulator of spermiogenesis in mice, a process during which sperm flagella are formed. The expression of Rc/btb2 is also regulated by multicilin, a protein that controls ciliogenesis. Given that mouse Rc/btb2 mRNA is not only expressed in tissues bearing motile cilia, but also in tissues without motile cilia, we investigated whether RC/BTB2 plays a role in the general process of ciliogenesis by studying two somatic cells lines that have primary cilia, NIH3T3 and IMCD3. We discovered that the subcellular localization of RT/BTB2 in the NIH3T3 and IMCD3 cells encompasses the pathway for ciliogenesis. RC/BTB2 was found in the Golgi bodies and centrosomes, two key structures essential for normal ciliogenesis. Knockdown of Rc/btb2 gene expression in these cell lines disrupted ciliogenesis. The percentage of cells with primary cilia was significantly reduced in stable cell lines transduced with specific Rc/btb2 shRNA viruses compared to the control cells. When cilia were formed in the knockdown cells, they were significantly shorter than those in the control cells. Knockdown of Rc/btb2 expression did not affect cell proliferation and the cell cycle. Exogenous expression of RC/BTB2 in these stable knockdown cells restored ciliogenesis. These findings suggest that RC/BTB2 is a necessary component of the process of formation of primary cilia in somatic cells, perhaps through the transportation of cargos from Golgi bodies to centrosomes for cilia assembling. PMID:25762510

  4. An Essential Role for Dermal Primary Cilia in Hair Follicle Morphogenesis

    PubMed Central

    Lehman, Jonathan; Laag, Essam; Michaud, Edward J.; Yoder, Bradley K.

    2009-01-01

    The primary cilium is a microtubule-based organelle implicated as an essential component of a number of signaling pathways. It is present on cells throughout the mammalian body; however, its functions in most tissues remain largely unknown. Herein we demonstrate that primary cilia are present on cells in murine skin and hair follicles throughout morphogenesis and during hair follicle cycling in postnatal life. Using the Cre-lox system, we disrupted cilia assembly in the ventral dermis and evaluated the effects on hair follicle development. Mice with disrupted dermal cilia have severe hypotrichosis (lack of hair) in affected areas. Histological analyses reveal that most follicles in the mutants arrest at stage 2 of hair development and have small or absent dermal condensates. This phenotype is reminiscent of that seen in the skin of mice lacking Shh or Gli2. In situ hybridization and quantitative RT-PCR analysis indicates that the hedgehog pathway is downregulated in the dermis of the cilia mutant hair follicles. Thus, these data establish cilia as a critical signaling component required for normal hair morphogenesis and suggest that this organelle is needed on cells in the dermis for reception of signals such as sonic hedgehog. PMID:18987668

  5. Loss of primary cilia upregulates renal hypertrophic signaling and promotes cystogenesis.

    PubMed

    Bell, P Darwin; Fitzgibbon, Wayne; Sas, Kelli; Stenbit, Antine E; Amria, May; Houston, Amber; Reichert, Ryan; Gilley, Sandra; Siegal, Gene P; Bissler, John; Bilgen, Mehmet; Chou, Peter Cheng-te; Guay-Woodford, Lisa; Yoder, Brad; Haycraft, Courtney J; Siroky, Brian

    2011-05-01

    Primary cilia dysfunction alters renal tubular cell proliferation and differentiation and associates with accelerated cyst formation in polycystic kidney disease. However, the mechanism leading from primary ciliary dysfunction to renal cyst formation is unknown. We hypothesize that primary cilia prevent renal cyst formation by suppressing pathologic tubular cell hypertrophy and proliferation. Unilateral nephrectomy initiates tubular cell hypertrophy and proliferation in the contralateral kidney and provides a tool to examine primary cilia regulation of renal hypertrophy. Conditional knockout of the primary cilia ift88 gene leads to delayed, adult-onset renal cystic disease, which provides a window of opportunity to conduct unilateral nephrectomy and examine downstream kinetics of renal hypertrophy and cyst formation. In wild-type animals, unilateral nephrectomy activated the mTOR pathway and produced appropriate structural and functional hypertrophy without renal cyst formation. However, in ift88 conditional knockout animals, unilateral nephrectomy triggered increased renal hypertrophy and accelerated renal cyst formation, leading to renal dysfunction. mTOR signaling also increased compared with wild-type animals, suggesting a mechanistic cascade starting with primary ciliary dysfunction, leading to excessive mTOR signaling and renal hypertrophic signaling and culminating in cyst formation. These data suggest that events initiating hypertrophic signaling, such as structural or functional loss of renal mass, may accelerate progression of adult polycystic kidney disease toward end-stage renal disease. PMID:21493775

  6. Primary cilia expression in bone marrow in response to mechanical stimulation in explant bioreactor culture.

    PubMed

    Coughlin, T R; Schiavi, J; Alyssa Varsanik, M; Voisin, M; Birmingham, E; Haugh, M G; McNamara, L M; Niebur, G L

    2016-01-01

    Bone marrow contains a multitude of mechanically sensitive cells that may participate in mechanotransduction. Primary cilia are sensory organelles expressed on mesenchymal stem cells (MSCs), osteoblasts, osteocytes, and other cell types that sense fluid flow in monolayer culture. In marrow, cilia could similarly facilitate the sensation of relative motion between adjacent cells or interstitial fluid. The goal of this study was to determine the response of cilia to mechanical stimulation of the marrow. Bioreactors were used to supply trabecular bone explants with low magnitude mechanical stimulation (LMMS) of 0.3 ×g at 30 Hz for 1 h/d, 5 d/week, inducing shear stresses in the marrow. Four groups were studied: unstimulated (UNSTIM), stimulated (LMMS), and with and without chloral hydrate (UNSTIM+CH and LMMS+CH, respectively), which was used to disrupt cilia. After 19 days of culture, immunohistochemistry for acetylated α-tubulin revealed that more cells expressed cilia in culture compared to in vivo controls. Stimulation decreased the number of cells expressing cilia in untreated explants, but not in CH-treated explants. MSCs represented a greater fraction of marrow cells in the untreated explants than CH-treated explants. MSCs harvested from the stimulated groups were more proliferative than in the unstimulated explants, but this effect was absent from CH treated explants. In contrast to the marrow, neither LMMS nor CH treatment affected bone formation as measured by mineralising surface. Computational models indicated that LMMS does not induce bone strain, and the reported effects were thus attributed to shear stress in the marrow. From a clinical perspective, genetic or pharmaceutical alterations of cilia expression may affect marrow health and function. PMID:27434268

  7. Hedgehog signaling regulates myelination in the peripheral nervous system through primary cilia.

    PubMed

    Yoshimura, Kentaro; Takeda, Sen

    2012-02-01

    Myelination is an essential prerequisite for the nervous system to transmit an impulse efficiently by a saltatory conduction. In the peripheral nervous system (PNS), Schwann cells (SCs) engage in myelination. However, a detailed molecular mechanism underlying myelination still remains unclear. In this study, we hypothesized that the primary cilia of SCs are the regulators of Hedgehog (Hh) signaling-mediated myelination. To confirm our hypothesis, we used mouse dorsal root ganglion (DRG)/SC co-cultures, wherein the behavior of SCs could be analyzed by maintaining the interaction of SCs with DRG neurons. Under these conditions, SCs had primary cilia, and Hh signaling molecules accumulated on the primary cilia. When the SCs were stimulated by the addition of desert hedgehog or smoothened agonist, formation of myelin segments on the DRG axons was facilitated. On the contrary, upon administration of cyclopamine, an inhibitor of Hh signaling, myelin segments became comparable to those of controls. Of note, the ratio of SCs harboring primary cilium reached the highest point during the early phase of myelination. Furthermore, the strongest effects of Hh on myelination were encountered during the same stage. These results collectively indicate that Hh signaling regulates myelin formation through primary cilia in the PNS. PMID:22101064

  8. 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

  9. 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. PMID:27161363

  10. Cyst growth, polycystins, and primary cilia in autosomal dominant polycystic kidney disease.

    PubMed

    Lee, Seung Hun; Somlo, Stefan

    2014-06-01

    The primary cilium of renal epithelia acts as a transducer of extracellular stimuli. Polycystin (PC)1 is the protein encoded by the PKD1 gene that is responsible for the most common and severe form of autosomal dominant polycystic kidney disease (ADPKD). PC1 forms a complex with PC2 via their respective carboxy-terminal tails. Both proteins are expressed in the primary cilia. Mutations in either gene affect the normal architecture of renal tubules, giving rise to ADPKD. PC1 has been proposed as a receptor that modulates calcium signals via the PC2 channel protein. The effect of PC1 dosage has been described as the rate-limiting modulator of cystic disease. Reduced levels of PC1 or disruption of the balance in PC1/PC2 level can lead to the clinical features of ADPKD, without complete inactivation. Recent data show that ADPKD resulting from inactivation of polycystins can be markedly slowed if structurally intact cilia are also disrupted at the same time. Despite the fact that no single model or mechanism from these has been able to describe exclusively the pathogenesis of cystic kidney disease, these findings suggest the existence of a novel cilia-dependent, cyst-promoting pathway that is normally repressed by polycystin function. The results enable us to rethink our current understanding of genetics and cilia signaling pathways of ADPKD. PMID:26877954

  11. GPR88 Reveals a Discrete Function of Primary Cilia as Selective Insulators of GPCR Cross-Talk

    PubMed Central

    Marley, Aaron; Choy, Regina Wai-Yan; von Zastrow, Mark

    2013-01-01

    A number of G protein-coupled receptors (GPCRs) localize to primary cilia but the functional significance of cilia to GPCR signaling remains incompletely understood. We investigated this question by focusing on the D1 dopamine receptor (D1R) and beta-2 adrenergic receptor (B2AR), closely related catecholamine receptors that signal by stimulating production of the diffusible second messenger cyclic AMP (cAMP) but differ in localization relative to cilia. D1Rs robustly concentrate on cilia of IMCD3 cells, as shown previously in other ciliated cell types, but disrupting cilia did not affect D1R surface expression or ability to mediate a concentration-dependent cAMP response. By developing a FRET-based biosensor suitable for resolving intra- from extra- ciliary cAMP changes, we found that the D1R-mediated cAMP response is not restricted to cilia and extends into the extra-ciliary cytoplasm. Conversely the B2AR, which we show here is effectively excluded from cilia, also generated a cAMP response in both ciliary and extra-ciliary compartments. We identified a distinct signaling effect of primary cilia through investigating GPR88, an orphan GPCR that is co-expressed with the D1R in brain, and which we show here is targeted to cilia similarly to the D1R. In ciliated cells, mutational activation of GPR88 strongly reduced the D1R-mediated cAMP response but did not affect the B2AR-mediated response. In marked contrast, in non-ciliated cells, GPR88 was distributed throughout the plasma membrane and inhibited the B2AR response. These results identify a discrete ‘insulating’ function of primary cilia in conferring selectivity on integrated catecholamine signaling through lateral segregation of receptors, and suggest a cellular activity of GPR88 that might underlie its effects on dopamine-dependent behaviors. PMID:23936473

  12. 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. PMID:26053317

  13. Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

    PubMed Central

    He, Qian; Wang, Guanghu; Wakade, Sushama; Dasgupta, Somsankar; Dinkins, Michael; Kong, Ji Na; Spassieva, Stefka D.; Bieberich, Erhard

    2014-01-01

    We show here that human embryonic stem (ES) and induced pluripotent stem cell–derived neuroprogenitors (NPs) develop primary cilia. Ciliogenesis depends on the sphingolipid ceramide and its interaction with atypical PKC (aPKC), both of which distribute to the primary cilium and the apicolateral cell membrane in NP rosettes. Neural differentiation of human ES cells to NPs is concurrent with a threefold elevation of ceramide—in particular, saturated, long-chain C16:0 ceramide (N-palmitoyl sphingosine) and nonsaturated, very long chain C24:1 ceramide (N-nervonoyl sphingosine). Decreasing ceramide levels by inhibiting ceramide synthase or neutral sphingomyelinase 2 leads to translocation of membrane-bound aPKC to the cytosol, concurrent with its activation and the phosphorylation of its substrate Aurora kinase A (AurA). Inhibition of aPKC, AurA, or a downstream target of AurA, HDAC6, restores ciliogenesis in ceramide-depleted cells. Of importance, addition of exogenous C24:1 ceramide reestablishes membrane association of aPKC, restores primary cilia, and accelerates neural process formation. Taken together, these results suggest that ceramide prevents activation of HDAC6 by cytosolic aPKC and AurA, which promotes acetylation of tubulin in primary cilia and, potentially, neural processes. This is the first report on the critical role of ceramide generated by nSMase2 in stem cell ciliogenesis and differentiation. PMID:24694597

  14. Primary cilia mechanosensing triggers autophagy-regulated cell volume control.

    PubMed

    Takacs, Zsuzsanna; Proikas-Cezanne, Tassula

    2016-05-27

    The primary cilium and the process of autophagy are thought to be in a functionally reciprocal relationship. In further support of this link, fluid flow sensing by the primary cilium is now shown to induce autophagy, which in turn regulates the volume of kidney epithelial cells. PMID:27230529

  15. Pulsed electromagnetic fields promote osteoblast mineralization and maturation needing the existence of primary cilia.

    PubMed

    Yan, Juan-Li; Zhou, Jian; Ma, Hui-Ping; Ma, Xiao-Ni; Gao, Yu-Hai; Shi, Wen-Gui; Fang, Qing-Qing; Ren, Qian; Xian, Cory J; Chen, Ke-Ming

    2015-03-15

    Although pulsed electromagnetic fields (PEMFs) have been approved as a therapy for osteoporosis, action mechanisms and optimal parameters are elusive. To determine the optimal intensity, exposure effects of 50 Hz PEMFs of 0.6-3.6 mT (0.6 interval at 90 min/day) were investigated on proliferation and osteogenic differentiation of cultured calvarial osteoblasts. All intensity groups stimulated proliferation significantly with the highest effect at 0.6 mT. The 0.6 mT group also obtained the optimal osteogenic effect as demonstrated by the highest ALP activity, ALP(+) CFU-f colony formation, nodule mineralization, and expression of COL-1 and BMP-2. To verify our hypothesis that the primary cilia are the cellular sensors for PEMFs, osteoblasts were also transfected with IFT88 siRNA or scrambled control, and osteogenesis-promoting effects of 0.6 mT PEMFs were found abrogated when primary cilia were inhibited by IFT88 siRNA. Thus primary cilia of osteoblasts play an indispensable role in mediating PEMF osteogenic effect in vitro. PMID:25661534

  16. Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development

    PubMed Central

    Bhattacharyya, Sohinee; Rainey, Mark A; Arya, Priyanka; Dutta, Samikshan; George, Manju; Storck, Matthew D.; McComb, Rodney D.; Muirhead, David; Todd, Gordon L.; Gould, Karen; Datta, Kaustubh; Waes, Janee Gelineau-van; Band, Vimla; Band, Hamid

    2016-01-01

    Members of the four-member C-terminal EPS15-Homology Domain-containing (EHD) protein family play crucial roles in endocytic recycling of cell surface receptors from endosomes to the plasma membrane. In this study, we show that Ehd1 gene knockout in mice on a predominantly B6 background is embryonic lethal. Ehd1-null embryos die at mid-gestation with a failure to complete key developmental processes including neural tube closure, axial turning and patterning of the neural tube. We found that Ehd1-null embryos display short and stubby cilia on the developing neuroepithelium at embryonic day 9.5 (E9.5). Loss of EHD1 also deregulates the ciliary SHH signaling with Ehd1-null embryos displaying features indicative of increased SHH signaling, including a significant downregulation in the formation of the GLI3 repressor and increase in the ventral neuronal markers specified by SHH. Using Ehd1-null MEFS we found that EHD1 protein co-localizes with the SHH receptor Smoothened in the primary cilia upon ligand stimulation. Under the same conditions, EHD1 was shown to co-traffic with Smoothened into the developing primary cilia and we identify EHD1 as a direct binding partner of Smoothened. Overall, our studies identify the endocytic recycling regulator EHD1 as a novel regulator of the primary cilium-associated trafficking of Smoothened and Hedgehog signaling. PMID:26884322

  17. Active stochastic stress fluctuations in the cell cytoskeleton stir the cell and activate primary cilia

    NASA Astrophysics Data System (ADS)

    Schmidt, Christoph F.; Fakhri, Nikta; Battle, Christopher; Ott, Carolyn M.; Wessel, Alok D.; Lippincott-Schwartz, Jennifer; Mackintosh, Frederick C.

    2015-03-01

    Cells are active systems with molecular force generation that drives complex dynamics at the supramolecular scale. Much of cellular dynamics is driven by myosin motors interacting with the actin cytoskeleton. We discovered active random ``stirring'' driven by cytoplasmic myosin as an intermediate mode of transport, different from both thermal diffusion and directed motor activity. We found a further manifestation of cytoskeletal dynamics in the active motion patterns of primary cilia generated by epithelial cells. These cilia were thought to be immotile due to the absence of dynein motors, but it turns out that their anchoring deeper inside the cell in combination with the strongly fluctuating cortex results in clearly measurable non-equilibrium fluctuations.

  18. Galectin-7 modulates the length of the primary cilia and wound repair in polarized kidney epithelial cells.

    PubMed

    Rondanino, Christine; Poland, Paul A; Kinlough, Carol L; Li, Hui; Rbaibi, Youssef; Myerburg, Michael M; Al-bataineh, Mohammad M; Kashlan, Ossama B; Pastor-Soler, Nuria M; Hallows, Kenneth R; Weisz, Ora A; Apodaca, Gerard; Hughey, Rebecca P

    2011-09-01

    Galectins (Gal) are β-galactoside-binding proteins that function in epithelial development and homeostasis. An overlapping role for Gal-3 and Gal-7 in wound repair was reported in stratified epithelia. Although Gal-7 was thought absent in simple epithelia, it was reported in a proteomic analysis of cilia isolated from cultured human airway, and we recently identified Gal-7 transcripts in Madin-Darby canine kidney (MDCK) cells (Poland PA, Rondanino C, Kinlough CL, Heimburg-Molinaro J, Arthur CM, Stowell SR, Smith DF, Hughey RP. J Biol Chem 286: 6780-6790, 2011). We now report that Gal-7 is localized exclusively on the primary cilium of MDCK, LLC-PK(1) (pig kidney), and mpkCCD(c14) (mouse kidney) cells as well as on cilia in the rat renal proximal tubule. Gal-7 is also present on most cilia of multiciliated cells in human airway epithelia primary cultures. Interestingly, exogenous glutathione S-transferase (GST)-Gal-7 bound the MDCK apical plasma membrane as well as the cilium, while the lectin Ulex europeaus agglutinin, with glycan preferences similar to Gal-7, bound the basolateral plasma membrane as well as the cilium. In pull-down assays, β1-integrin isolated from either the basolateral or apical/cilia membranes of MDCK cells was similarly bound by GST-Gal-7. Selective localization of Gal-7 to cilia despite the presence of binding sites on all cell surfaces suggests that intracellular Gal-7 is specifically delivered to cilia rather than simply binding to surface glycoconjugates after generalized secretion. Moreover, depletion of Gal-7 using tetracycline-induced short-hairpin RNA in mpkCCD(c14) cells significantly reduced cilia length and slowed wound healing in a scratch assay. We conclude that Gal-7 is selectively targeted to cilia and plays a key role in surface stabilization of glycoconjugates responsible for integrating cilia function with epithelial repair. PMID:21677144

  19. Axonemal Positioning and Orientation in 3-D Space for Primary Cilia: What is Known, What is Assumed, and What Needs Clarification

    PubMed Central

    Farnum, Cornelia E.; Wilsman, Norman J.

    2012-01-01

    Two positional characteristics of the ciliary axoneme – its location on the plasma membrane as it emerges from the cell, and its orientation in three-dimensional space – are known to be critical for optimal function of actively motile cilia (including nodal cilia), as well as for modified cilia associated with special senses. However, these positional characteristics have not been analyzed to any significant extent for primary cilia. This review briefly summarizes the history of knowledge of these two positional characteristics across a wide spectrum of cilia, emphasizing their importance for proper function. Then the review focuses what is known about these same positional characteristics for primary cilia in all major tissue types where they have been reported. The review emphasizes major areas that would be productive for future research for understanding how positioning and 3-D orientation of primary cilia may be related to their hypothesized signaling roles within different cellular populations. PMID:22012592

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

    PubMed

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

    2016-04-01

    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 commitmentin 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 cellsin 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 commitmentin vivoand 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

  1. Neonatal seizures induced by pentylenetetrazol or kainic acid disrupt primary cilia growth on developing mouse cortical neurons.

    PubMed

    Parker, Alexander K; Le, Megan M; Smith, Tyler S; Hoang-Minh, Lan B; Atkinson, Eric W; Ugartemendia, George; Semple-Rowland, Susan; Coleman, Jason E; Sarkisian, Matthew R

    2016-08-01

    Neonatal or early-life seizures (ELS) are often associated with life-long neurophysiological, cognitive and behavioral deficits, but the underlying mechanisms contributing to these deficits remain poorly understood. Newborn, post-migratory cortical neurons sprout ciliary buds (procilia) that mature into primary cilia. Disruption of the growth or signaling capabilities of these cilia has been linked to atypical neurite outgrowth from neurons and abnormalities in neuronal circuitry. Here, we tested the hypothesis that generalized seizures induced by pentylenetetrazol (PTZ) or kainic acid (KA) during early postnatal development impair neuronal and/or glial ciliogenesis. Mice received PTZ (50 or 100mg/kg), KA (2mg/kg), or saline either once at birth (P0), or once daily from P0 to P4. Using immunohistochemistry and electron microscopy, the cilia of neurons and glia were examined at P7, P14, and P42. A total of 83 regions were analyzed, representing 13 unique neocortical and hippocampal regions. Neuronal cilia were identified by co-expression of NeuN and type 3 adenylyl cyclase (ACIII) or somatostatin receptor 3 (SSTR3), while glial cilia were identified by co-expression of GFAP, Arl13b, and gamma-tubulin. We found that PTZ exposure at either P0 or from P0 to P4 induced convulsive behavior, followed by acute and lasting effects on neuronal cilia lengths that varied depending on the cortical region, PTZ dose, injection frequency, and time post-PTZ. Both increases and decreases in neuronal cilia length were observed. No changes in the length of glial cilia were observed under any of the test conditions. Lastly, we found that a single KA seizure at P0 led to similar abnormalities in neuronal cilia lengths. Our results suggest that seizure(s) occurring during early stages of cortical development induce persistent and widespread changes in neuronal cilia length. Given the impact neuronal cilia have on neuronal differentiation, ELS-induced changes in ciliogenesis may

  2. Phosphorylation-dependent Akt-Inversin interaction at the basal body of primary cilia.

    PubMed

    Suizu, Futoshi; Hirata, Noriyuki; Kimura, Kohki; Edamura, Tatsuma; Tanaka, Tsutomu; Ishigaki, Satoko; Donia, Thoria; Noguchi, Hiroko; Iwanaga, Toshihiko; Noguchi, Masayuki

    2016-06-15

    A primary cilium is a microtubule-based sensory organelle that plays an important role in human development and disease. However, regulation of Akt in cilia and its role in ciliary development has not been demonstrated. Using yeast two-hybrid screening, we demonstrate that Inversin (INVS) interacts with Akt. Mutation in the INVS gene causes nephronophthisis type II (NPHP2), an autosomal recessive chronic tubulointerstitial nephropathy. Co-immunoprecipitation assays show that Akt interacts with INVS via the C-terminus. In vitro kinase assays demonstrate that Akt phosphorylates INVS at amino acids 864-866 that are required not only for Akt interaction, but also for INVS dimerization. Co-localization of INVS and phosphorylated form of Akt at the basal body is augmented by PDGF-AA Akt-null MEF cells as well as siRNA-mediated inhibition of Akt attenuated ciliary growth, which was reversed by Akt reintroduction. Mutant phosphodead- or NPHP2-related truncated INVS, which lack Akt phosphorylation sites, suppress cell growth and exhibit distorted lumen formation and misalignment of spindle axis during cell division. Further studies will be required for elucidating functional interactions of Akt-INVS at the primary cilia for identifying the molecular mechanisms underlying NPHP2. PMID:27220846

  3. Primary cilia function regulates the length of the embryonic trunk axis and urogenital field in mice.

    PubMed

    Wainwright, Elanor N; Svingen, Terje; Ng, Ee Ting; Wicking, Carol; Koopman, Peter

    2014-11-15

    The issues of whether and how some organs coordinate their size and shape with the blueprint of the embryo axis, while others appear to regulate their morphogenesis autonomously, remain poorly understood. Mutations in Ift144, encoding a component of the trafficking machinery of primary cilia assembly, result in a range of embryo patterning defects, affecting the limbs, skeleton and neural system. Here, we show that embryos of the mouse mutant Ift144(twt) develop gonads that are larger than wild-type. Investigation of the early patterning of the urogenital ridge revealed that the anterior-posterior domain of the gonad/mesonephros was extended at 10.5 dpc, with no change in the length of the metanephros. In XY embryos, this extension resulted in an increase in testis cord number. Moreover, we observed a concomitant extension of the trunk axis in both sexes, with no change in the length of the tail domain or somite number. Our findings support a model in which: (1) primary cilia regulate embryonic trunk elongation; (2) the length of the trunk axis determines the size of the urogenital ridges; and (3) the gonad domain is partitioned into a number of testis cords that depends on the available space, rather than being divided a predetermined number of times to generate a specific number of cords. PMID:25224227

  4. 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. PMID:27435857

  5. Meckel-Gruber syndrome and the role of primary cilia in kidney, skeleton, and central nervous system development

    PubMed Central

    Barker, Amy R; Thomas, Rhys; Dawe, Helen R

    2014-01-01

    The ciliopathies are a group of related inherited diseases characterized by malformations in organ development. The diseases affect multiple organ systems, with kidney, skeleton, and brain malformations frequently observed. Research over the last decade has revealed that these diseases are due to defects in primary cilia, essential sensory organelles found on most cells in the human body. Here we discuss the genetic and cell biological basis of one of the most severe ciliopathies, Meckel-Gruber syndrome, and explain how primary cilia contribute to the development of the affected organ systems. PMID:24322779

  6. Primary cilia distribution and orientation during involution of the bovine mammary gland.

    PubMed

    Biet, J; Poole, C A; Stelwagen, K; Margerison, J K; Singh, K

    2016-05-01

    The regulation of mammary gland involution occurs through multiple levels including environmental factors, hormones, and local intramammary signals. Primary cilia (PC) are signaling organelles that sense biochemical and biophysical extracellular stimuli and are vital for cellular and tissue function. The aim of this study was to examine the distribution, incidence, and orientation of PC. Furthermore, we determined changes in expression levels of the signal transducer and activator of transcription (STAT)6 at the onset of bovine mammary gland involution. Mammary tissue was collected from pasture-fed, primiparous, nonpregnant Friesian dairy cows at mid lactation (n=5 per group) killed 6-h after milking (lactating controls) and during involution after 7 and 28 d of nonmilking (NM). Fluorescent immunohistochemistry and confocal microscopy of tissue sections showed that PC were present on luminal secretory epithelial cells (SEC), myoepithelial cells (MEC), and stromal fibroblast cells (SFC). Furthermore, in all 3 experimental groups, different PC positions or orientations relative to the cell surface were identified on SEC and MEC, which projected toward the lumen and were either straight, bent, or deflected against the apical cell surface, whereas PC in SFC were confined to the interalveolar space. However, by 28-d NM, fewer PC projected into the luminal space and most appeared deflected or projected toward the interalveolar space. Furthermore, by 28-d NM, with the increase in stromal connective tissue, more PC were detected within the interalveolar and interlobular stroma. At 28-d NM, we observed a decrease in luminal cilia relative to the total number of cilia. The number of ciliated cells in the total fraction (SEC, MEC, and SFC) was the same for all 3 groups, although in the luminal fraction (SEC and MEC), PC per nuclei increased by 28-d NM relative to lactation. At all 3 stages, we detected variations in shape and orientation of PC within the same alveolus, with

  7. RTTN Mutations Link Primary Cilia Function to Organization of the Human Cerebral Cortex

    PubMed Central

    Kheradmand Kia, Sima; Verbeek, Elly; Engelen, Erik; Schot, Rachel; Poot, Raymond A.; de Coo, Irenaeus F.M.; Lequin, Maarten H.; Poulton, Cathryn J.; Pourfarzad, Farzin; Grosveld, Frank G.; Brehm, António; de Wit, Marie Claire Y.; Oegema, Renske; Dobyns, William B.; Verheijen, Frans W.; Mancini, Grazia M.S.

    2012-01-01

    Polymicrogyria is a malformation of the developing cerebral cortex caused by abnormal organization and characterized by many small gyri and fusion of the outer molecular layer. We have identified autosomal-recessive mutations in RTTN, encoding Rotatin, in individuals with bilateral diffuse polymicrogyria from two separate families. Rotatin determines early embryonic axial rotation, as well as anteroposterior and dorsoventral patterning in the mouse. Human Rotatin has recently been identified as a centrosome-associated protein. The Drosophila melanogaster homolog of Rotatin, Ana3, is needed for structural integrity of centrioles and basal bodies and maintenance of sensory neurons. We show that Rotatin colocalizes with the basal bodies at the primary cilium. Cultured fibroblasts from affected individuals have structural abnormalities of the cilia and exhibit downregulation of BMP4, WNT5A, and WNT2B, which are key regulators of cortical patterning and are expressed at the cortical hem, the cortex-organizing center that gives rise to Cajal-Retzius (CR) neurons. Interestingly, we have shown that in mouse embryos, Rotatin colocalizes with CR neurons at the subpial marginal zone. Knockdown experiments in human fibroblasts and neural stem cells confirm a role for RTTN in cilia structure and function. RTTN mutations therefore link aberrant ciliary function to abnormal development and organization of the cortex in human individuals. PMID:22939636

  8. Primary ciliary dyskinesia: evaluation using cilia beat frequency assessment via spectral analysis of digital microscopy images

    PubMed Central

    Kögler, João E.; Macchione, Mariangela; Shoemark, Amelia; Saldiva, Paulo H. N.; Rodrigues, Joaquim C.

    2011-01-01

    Ciliary beat frequency (CBF) measurements provide valuable information for diagnosing of primary ciliary dyskinesia (PCD). We developed a system for measuring CBF, used it in association with electron microscopy to diagnose PCD, and then analyzed characteristics of PCD patients.1 The CBF measurement system was based on power spectra measured through digital imaging. Twenty-four patients suspected of having PCD (age 1–19 yr) were selected from a group of 75 children and adolescents with pneumopathies of unknown causes. Ten healthy, nonsmoking volunteers (age ≥17 yr) served as a control group. Nasal brush samples were collected, and CBF and electron microscopy were performed. PCD was diagnosed in 12 patients: 5 had radial spoke defects, 3 showed absent central microtubule pairs with transposition, 2 had outer dynein arm defects, 1 had a shortened outer dynein arm, and 1 had a normal ultrastructure. Previous studies have reported that the most common cilia defects are in the dynein arm. As expected, the mean CBF was higher in the control group (P < 0.001) and patients with normal ultrastructure (P < 0.002), than in those diagnosed with cilia ultrastructural defects (i.e., PCD patients). An obstructive ventilatory pattern was observed in 70% of the PCD patients who underwent pulmonary function tests. All PCD patients presented bronchial wall thickening on chest computed tomography scans. The protocol and diagnostic techniques employed allowed us to diagnose PCD in 16% of patients in this study. PMID:21551013

  9. Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1

    PubMed Central

    Milenkovic, Ljiljana; Weiss, Lucien E.; Yoon, Joshua; Roth, Theodore L.; Su, YouRong S.; Sahl, Steffen J.; Scott, Matthew P.; Moerner, W. E.

    2015-01-01

    Accumulation of the signaling protein Smoothened (Smo) in the membrane of primary cilia is an essential step in Hedgehog (Hh) signal transduction, yet the molecular mechanisms of Smo movement and localization are poorly understood. Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation. PMID:26100903

  10. Primary cilia act as mechanosensors during bone healing around an implant.

    PubMed

    Leucht, P; Monica, S D; Temiyasathit, S; Lenton, K; Manu, A; Longaker, M T; Jacobs, C R; Spilker, R L; Guo, H; Brunski, J B; Helms, J A

    2013-03-01

    The primary cilium is an organelle that senses cues in a cell's local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3a(fl/fl) mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective. PMID:22784673

  11. Primary cilia act as mechanosensors during bone healing around an implant

    PubMed Central

    Leucht, P.; Monica, S.D.; Temiyasathit, S.; Lenton, K.; Manu, A.; Longaker, M.T.; Jacobs, C.R.; Spilker, R.L.; Guo, H.; Brunski, J.B.; Helms, J.A.

    2012-01-01

    The primary cilium is an organelle that senses cues in a cell’s local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3afl/fl mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective. PMID:22784673

  12. Biological and Chemical Removal of Primary Cilia Affects Mechanical Activation of Chondrogenesis Markers in Chondroprogenitors and Hypertrophic Chondrocytes

    PubMed Central

    Deren, Matthew E.; Yang, Xu; Guan, Yingjie; Chen, Qian

    2016-01-01

    Chondroprogenitors and hypertrophic chondrocytes, which are the first and last stages of the chondrocyte differentiation process, respectively, are sensitive to mechanical signals. We hypothesize that the mechanical sensitivity of these cells depends on the cell surface primary cilia. To test this hypothesis, we removed the primary cilia by biological means with transfection with intraflagellar transport protein 88 (IFT88) siRNA or by chemical means with chloral hydrate treatment. Transfection of IFT88 siRNA significantly reduced the percentage of ciliated cells in both chondroprogenitor ATDC5 cells as well as primary hypertrophic chondrocytes. Cyclic loading (1 Hz, 10% matrix deformation) of ATDC5 cells in three-dimensional (3D) culture stimulates the mRNA levels of chondrogenesis marker Type II collagen (Col II), hypertrophic chondrocyte marker Type X collagen (Col X), and a molecular regulator of chondrogenesis and chondrocyte hypertrophy bone morphogenetic protein 2 (BMP-2). The reduction of ciliated chondroprogenitors abolishes mechanical stimulation of Col II, Col X, and BMP-2. In contrast, cyclic loading stimulates Col X mRNA levels in hypertrophic chondrocytes, but not those of Col II and BMP-2. Both biological and chemical reduction of ciliated hypertrophic chondrocytes reduced but failed to abolish mechanical stimulation of Col X mRNA levels. Thus, primary cilia play a major role in mechanical stimulation of chondrogenesis and chondrocyte hypertrophy in chondroprogenitor cells and at least a partial role in hypertrophic chondrocytes. PMID:26861287

  13. Primary cilia regulate mTORC1 activity and cell size through Lkb1.

    PubMed

    Boehlke, Christopher; Kotsis, Fruzsina; Patel, Vishal; Braeg, Simone; Voelker, Henriette; Bredt, Saskia; Beyer, Theresa; Janusch, Heike; Hamann, Christoph; Gödel, Markus; Müller, Klaus; Herbst, Martin; Hornung, Miriam; Doerken, Mara; Köttgen, Michael; Nitschke, Roland; Igarashi, Peter; Walz, Gerd; Kuehn, E Wolfgang

    2010-11-01

    The mTOR pathway is the central regulator of cell size. External signals from growth factors and nutrients converge on the mTORC1 multi-protein complex to modulate downstream targets, but how the different inputs are integrated and translated into specific cellular responses is incompletely understood. Deregulation of the mTOR pathway occurs in polycystic kidney disease (PKD), where cilia (filiform sensory organelles) fail to sense urine flow because of inherited mutations in ciliary proteins. We therefore investigated if cilia have a role in mTOR regulation. Here, we show that ablation of cilia in transgenic mice results in enlarged cells when compared with control animals. In vitro analysis demonstrated that bending of the cilia by flow is required for mTOR downregulation and cell-size control. Surprisingly, regulation of cell size by cilia is independent of flow-induced calcium transients, or Akt. However, the tumour-suppressor protein Lkb1 localises in the cilium, and flow results in increased AMPK phosphorylation at the basal body. Conversely, knockdown of Lkb1 prevents normal cell-size regulation under flow conditions. Our results demonstrate that the cilium regulates mTOR signalling and cell size, and identify the cilium-basal body compartment as a spatially restricted activation site for Lkb1 signalling. PMID:20972424

  14. Functional aspects of primary cilia in signaling, cell cycle and tumorigenesis

    PubMed Central

    2013-01-01

    Dysfunctional cilia underlie a broad range of cellular and tissue phenotypes and can eventually result in the development of ciliopathies: pathologically diverse diseases that range from clinically mild to highly complex and severe multi-organ failure syndromes incompatible with neonatal life. Given that virtually all cells of the human body have the capacity to generate cilia, it is likely that clinical manifestations attributed to ciliary dysfunction will increase in the years to come. Disputed but nevertheless enigmatic is the notion that at least a subset of tumor phenotypes fit within the ciliopathy disease spectrum and that cilia loss may be required for tumor progression. Contending for the centrosome renders ciliation and cell division mutually exclusive; a regulated tipping of balance promotes either process. The mechanisms involved, however, are complex. If the hypothesis that tumorigenesis results from dysfunctional cilia is true, then why do the classic ciliopathies only show limited hyperplasia at best? Although disassembly of the cilium is a prerequisite for cell proliferation, it does not intrinsically drive tumorigenesis per se. Alternatively, we will explore the emerging evidence suggesting that some tumors depend on ciliary signaling. After reviewing the structure, genesis and signaling of cilia, the various ciliopathy syndromes and their genetics, we discuss the current debate of tumorigenesis as a ciliopathy spectrum defect, and describe recent advances in this fascinating field. PMID:23628112

  15. Kinetics of Hedgehog-Dependent Full-Length Gli3 Accumulation in Primary Cilia and Subsequent Degradation ▿ † ‡

    PubMed Central

    Wen, Xiaohui; Lai, Cary K.; Evangelista, Marie; Hongo, Jo-Anne; de Sauvage, Frederic J.; Scales, Suzie J.

    2010-01-01

    Hedgehog (Hh) signaling in vertebrates depends on intraflagellar transport (IFT) within primary cilia. The Hh receptor Patched is found in cilia in the absence of Hh and is replaced by the signal transducer Smoothened within an hour of Hh stimulation. By generating antibodies capable of detecting endogenous pathway transcription factors Gli2 and Gli3, we monitored their kinetics of accumulation in cilia upon Hh stimulation. Localization occurs within minutes of Hh addition, making it the fastest reported readout of pathway activity, which permits more precise temporal and spatial localization of Hh signaling events. We show that the species of Gli3 that accumulates at cilium tips is full-length and likely not protein kinase A phosphorylated. We also confirmed that phosphorylation and βTrCP/Cul1 are required for endogenous Gli3 processing and that this is inhibited by Hh. Surprisingly, however, Hh-dependent inhibition of processing does not lead to accumulation of full-length Gli3, but instead renders it labile, leading to its proteasomal degradation via the SPOP/Cul3 complex. In fact, full-length Gli3 disappears with faster kinetics than the Gli3 repressor, the latter not requiring SPOP/Cul3 or βTrCP/Cul1. This may contribute to the increased Gli3 activator/repressor ratios found in IFT mutants. PMID:20154143

  16. EFCAB7 and IQCE regulate Hedgehog signaling by tethering the EVC-EVC2 complex to the base of primary cilia

    PubMed Central

    Pusapati, Ganesh V.; Hughes, Casey E; Dorn, Karolin V.; Zhang, Dapeng; Sugianto, Priscilla; Aravind, L.; Rohatgi, Rajat

    2014-01-01

    The Hedgehog (Hh) pathway depends on primary cilia in vertebrates, but the signaling machinery within cilia remains incompletely defined. We report the identification of a complex between two ciliary proteins, EFCAB7 and IQCE, which positively regulates the Hh pathway. The EFCAB7-IQCE module anchors the EVC-EVC2 complex in a signaling microdomain at the base of cilia. EVC and EVC2 genes are mutated in Ellis van Creveld and Weyers syndromes, characterized by impaired Hh signaling in skeletal, cardiac and orofacial tissues. EFCAB7 binds to a C-terminal disordered region in EVC2 that is deleted in Weyers patients. EFCAB7 depletion mimics the Weyers cellular phenotype— the mis-localization of EVC-EVC2 within cilia and impaired activation of the transcription factor GLI2. Evolutionary analysis suggests that emergence of these complexes might have been important for adaptation of an ancient organelle, the cilium, for an animal-specific signaling network. PMID:24582806

  17. Concise Review: Primary Cilia: Control Centers for Stem Cell Lineage Specification and Potential Targets for Cell-Based Therapies.

    PubMed

    Bodle, Josephine C; Loboa, Elizabeth G

    2016-06-01

    Directing stem cell lineage commitment prevails as the holy grail of translational stem cell research, particularly to those interested in the application of mesenchymal stem cells and adipose-derived stem cells in tissue engineering. However, elucidating the mechanisms underlying their phenotypic specification persists as an active area of research. In recent studies, the primary cilium structure has been intimately associated with defining cell phenotype, maintaining stemness, as well as functioning in a chemo, electro, and mechanosensory capacity in progenitor and committed cell types. Many hypothesize that the primary cilium may indeed be another important player in defining and controlling cell phenotype, concomitant with lineage-dictated cytoskeletal dynamics. Many of the studies on the primary cilium have emerged from disparate areas of biological research, and crosstalk amongst these areas of research is just beginning. To date, there has not been a thorough review of how primary cilia fit into the current paradigm of stem cell differentiation and this review aims to summarize the current cilia work in this context. The goal of this review is to highlight the cilium's function and integrate this knowledge into the working knowledge of stem cell biologists and tissue engineers developing regenerative medicine technologies. Stem Cells 2016;34:1445-1454. PMID:26866419

  18. Inv acts as a molecular anchor for Nphp3 and Nek8 in the proximal segment of primary cilia.

    PubMed

    Shiba, Dai; Manning, Danielle K; Koga, Hisashi; Beier, David R; Yokoyama, Takahiko

    2010-02-01

    A primary cilium is an antenna-like structure extending from the surface of most vertebrate cells. It is structurally divided along its vertical axis into sub-compartments that include the ciliary tip, the shaft, the ciliary necklace segment, the transitional zone and the basal body. We recently discovered that the shaft of the primary cilia has a distinct molecular compartment, termed the "Inv compartment", which is characterized by the accumulation of Inv at the base of primary cilia. Inv was discovered as a causative gene in inv mutant mice. It was later found to be responsible for the infantile type of nephronophthisis (NPHP2). Nephronophthisis (NPHP) is an autosomal recessive kidney disease. Nine causative genes have been identified, with all examined products thought to function in cilia, basal body and/or centrioles. However, their exact intra-ciliary localization and relationship have not been clear. Here, we report that products of Nphp3 and Nek8 (the mouse orthologs of the causative genes for NPHP3 and NPHP9, respectively) localize to the Inv compartment. We also show that Inv is essential for the compartmental localization of Nphp3 and Nek8, whereas localization of Inv does not require Nphp3 or Nek8. Nphp1 and Nphp4 also localize at the proximal region of the cilium, but not in Inv compartment. Our results indicate that Inv acts as an anchor for Nphp3 and Nek8 in the Inv compartment, and suggest that Inv compartment is a candidate site for intra-ciliary interaction of Inv, Nphp3 and Nek8. PMID:20169535

  19. Shear stress blunts tubuloglomerular feedback partially mediated by primary cilia and nitric oxide at the macula densa.

    PubMed

    Wang, Lei; Shen, Chunyu; Liu, Haifeng; Wang, Shaohui; Chen, Xinshan; Roman, Richard J; Juncos, Luis A; Lu, Yan; Wei, Jin; Zhang, Jie; Yip, Kay-Pong; Liu, Ruisheng

    2015-10-01

    The present study tested whether primary cilia on macula densa serve as a flow sensor to enhance nitric oxide synthase 1 (NOS1) activity and inhibit tubuloglomerular feedback (TGF). Isolated perfused macula densa was loaded with calcein red and 4,5-diaminofluorescein diacetate to monitor cell volume and nitric oxide (NO) generation. An increase in tubular flow rate from 0 to 40 nl/min enhanced NO production by 40.0 ± 1.2%. The flow-induced NO generation was blocked by an inhibitor of NOS1 but not by inhibition of the Na/K/2Cl cotransporter or the removal of electrolytes from the perfusate. NO generation increased from 174.8 ± 21 to 276.1 ± 24 units/min in cultured MMDD1 cells when shear stress was increased from 0.5 to 5.0 dynes/cm(2). The shear stress-induced NO generation was abolished in MMDD1 cells in which the cilia were disrupted using a siRNA to ift88. Increasing the NaCl concentration of the tubular perfusate from 10 to 80 mM NaCl in the isolated perfused juxtaglomerular preparation reduced the diameter of the afferent arteriole by 3.8 ± 0.1 μm. This response was significantly blunted to 2.5 ± 0.2 μm when dextran was added to the perfusate to increase the viscosity and shear stress. Inhibition of NOS1 blocked the effect of dextran on TGF response. In vitro, the effects of raising perfusate viscosity with dextran on tubular hydraulic pressure were minimized by reducing the outflow resistance to avoid stretching of tubular cells. These results suggest that shear stress stimulates primary cilia on the macula densa to enhance NO generation and inhibit TGF responsiveness. PMID:26269519

  20. Planar cell polarity effector gene Intu regulates cell fate-specific differentiation of keratinocytes through the primary cilia.

    PubMed

    Dai, D; Li, L; Huebner, A; Zeng, H; Guevara, E; Claypool, D J; Liu, A; Chen, J

    2013-01-01

    Genes involved in the planar cell polarity (PCP) signaling pathway are essential for a number of developmental processes in mammals, such as convergent extension and ciliogenesis. Tissue-specific PCP effector genes of the PCP signaling pathway are believed to mediate PCP signals in a tissue- and cell type-specific manner. However, how PCP signaling controls the morphogenesis of mammalian tissues remains unclear. In this study, we investigated the role of inturned (Intu), a tissue-specific PCP effector gene, during hair follicle formation in mice. Tissue-specific disruption of Intu in embryonic epidermis resulted in hair follicle morphogenesis arrest because of the failure of follicular keratinocyte to differentiate. Targeting Intu in the epidermis resulted in almost complete loss of primary cilia in epidermal and follicular keratinocytes, and a suppressed hedgehog signaling pathway. Surprisingly, the epidermal stratification and differentiation programs and barrier function were not affected. These results demonstrate that tissue-specific PCP effector genes of the PCP signaling pathway control the differentiation of keratinocytes through the primary cilia in a cell fate- and context-dependent manner, which may be critical in orchestrating the propagation and interpretation of polarity signals established by the core PCP components. PMID:22935613

  1. Changes in cell surface primary cilia and microvilli concurrent with measurements of fluid flow across the rabbit corneal endothelium ex vivo.

    PubMed

    Doughty, M J

    1998-12-01

    Primary cilia and microvilli have been reported on the mammalian rabbit corneal endothelium but their relationship to cell function is undefined. Six corneas from healthy 2 kg female albino rabbits were glutaraldehyde-fixed post mortem (15:00 h) or twelve corneal stroma-endothelial preparations incubated at 37 degrees C under an applied hydrostatic pressure of 20 cm H2O for 4 h prior to fixation. The corneal endothelium was assessed by quantitative scanning electron microscopy. Cells fixed immediately post mortem were decorated with small stubby microvilli (average 21 +/- 13/100 micron 2), and only 25% of the cells were decorated with primary cilia having an average length of 2.44 +/- 1.56 microns. Following 4 h ex vivo incubation with a phosphate-buffered Ringer solution, conspicuous microvilli developed to an average density of 40 +/- 19/100 micron 2 and primary cilia were found on 12% of the cells, having on average length of 2.27 +/- 1.38 microns. Following 4 h incubation in a bicarbonate-buffered Ringer solution, small stubby microvilli developed to a density of 49 +/- 18/100 micron 2, and 40% of the cells showed primary cilia with an average length of 4.31 +/- 1.93 microns; the net trans-endothelial fluid flow in the latter set was 60% greater. These studies indicate that the primary cilia on corneal endothelial cells might be responsive to fluid flow, but that mild mechanical and/or chemical stress could also be the cause of the change since the elaboration of primary cilia can be accompanied by microvilli as well. PMID:10036788

  2. Cilia dysfunction in lung disease.

    PubMed

    Tilley, Ann E; Walters, Matthew S; Shaykhiev, Renat; Crystal, Ronald G

    2015-01-01

    A characteristic feature of the human airway epithelium is the presence of ciliated cells bearing motile cilia, specialized cell surface projections containing axonemes composed of microtubules and dynein arms, which provide ATP-driven motility. In the airways, cilia function in concert with airway mucus to mediate the critical function of mucociliary clearance, cleansing the airways of inhaled particles and pathogens. The prototypical disorder of respiratory cilia is primary ciliary dyskinesia, an inherited disorder that leads to impaired mucociliary clearance, to repeated chest infections, and to the progressive destruction of lung architecture. Numerous acquired lung diseases are also marked by abnormalities in both cilia structure and function. In this review we summarize current knowledge regarding airway ciliated cells and cilia, how they function to maintain a healthy epithelium, and how disorders of cilia structure and function contribute to inherited and acquired lung disease. PMID:25386990

  3. Cilia Dysfunction in Lung Disease

    PubMed Central

    Tilley, Ann E.; Walters, Matthew S.; Shaykhiev, Renat; Crystal, Ronald G.

    2015-01-01

    A characteristic feature of the human airway epithelium is the presence of ciliated cells bearing motile cilia, specialized cell surface projections containing axonemes comprised of microtubules and dynein arms, which provide ATP-driven motility. In the airways, cilia function in concert with airway mucus to mediate the critical function of mucociliary clearance, cleansing the airways of inhaled particles and pathogens. The prototypical disorder of respiratory cilia is primary ciliary dyskinesia, an inherited disorder that leads to impaired mucociliary clearance, repeated chest infections, and progressive destruction of lung architecture. Numerous acquired lung diseases are also marked by abnormalities in both cilia structure and function. In this review we summarize current knowledge regarding airway ciliated cells and cilia, how they function to maintain a healthy epithelium, and how disorders of cilia structure and function contribute to inherited and acquired lung disease. PMID:25386990

  4. PTEN regulates cilia through Dishevelled

    PubMed Central

    Shnitsar, Iryna; Bashkurov, Mikhail; Masson, Glenn R.; Ogunjimi, Abiodun A.; Mosessian, Sherly; Cabeza, Eduardo Aguiar; Hirsch, Calley L.; Trcka, Daniel; Gish, Gerald; Jiao, Jing; Wu, Hong; Winklbauer, Rudolf; Williams, Roger L.; Pelletier, Laurence; Wrana, Jeffrey L.; Barrios-Rodiles, Miriam

    2015-01-01

    Cilia are hair-like cellular protrusions important in many aspects of eukaryotic biology. For instance, motile cilia enable fluid movement over epithelial surfaces, while primary (sensory) cilia play roles in cellular signalling. The molecular events underlying cilia dynamics, and particularly their disassembly, are not well understood. Phosphatase and tensin homologue (PTEN) is an extensively studied tumour suppressor, thought to primarily act by antagonizing PI3-kinase signalling. Here we demonstrate that PTEN plays an important role in multicilia formation and cilia disassembly by controlling the phosphorylation of Dishevelled (DVL), another ciliogenesis regulator. DVL is a central component of WNT signalling that plays a role during convergent extension movements, which we show here are also regulated by PTEN. Our studies identify a novel protein substrate for PTEN that couples PTEN to regulation of cilia dynamics and WNT signalling, thus advancing our understanding of potential underlying molecular etiologies of PTEN-related pathologies. PMID:26399523

  5. NIMA-related kinases defective in murine models of polycystic kidney diseases localize to primary cilia and centrosomes.

    PubMed

    Mahjoub, Moe R; Trapp, Melissa L; Quarmby, Lynne M

    2005-12-01

    A key feature of the polycystic kidney diseases is aberrant cell proliferation, a consequence of dysfunctional ciliary signaling. The NIMA-related kinases (Nek) Nek1 and Nek8 carry the causal mutations of two of the eight established mouse models of polycystic kidneys. Nek proteins have roles in cell cycle and may contribute to coordinate regulation of cilia and cell-cycle progression. Herein is reported that in a mouse kidney epithelial cell line, mNek1 localizes to centrosomes in interphase and remains associated with the mitotic spindle pole during mitosis. In contrast, mNek8 localizes to the proximal region of the primary cilium and is not observed in dividing cells. Knockdown of mNek8 by siRNA does not affect ciliary assembly. Taken together with the phenotypes of the mutant mice, these data suggest that mNek1 and mNek8 provide links between cilia, centrosomes, and cell-cycle regulation. PMID:16267153

  6. Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse.

    PubMed

    Coelho, Paula A; Bury, Leah; Shahbazi, Marta N; Liakath-Ali, Kifayathullah; Tate, Peri H; Wormald, Sam; Hindley, Christopher J; Huch, Meritxell; Archer, Joy; Skarnes, William C; Zernicka-Goetz, Magdalena; Glover, David M

    2015-12-01

    To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development. PMID:26701933

  7. Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse

    PubMed Central

    Coelho, Paula A.; Bury, Leah; Shahbazi, Marta N.; Liakath-Ali, Kifayathullah; Tate, Peri H.; Wormald, Sam; Hindley, Christopher J.; Huch, Meritxell; Archer, Joy; Skarnes, William C.; Zernicka-Goetz, Magdalena; Glover, David M.

    2015-01-01

    To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development. PMID:26701933

  8. Mutations in INVS encoding inversin cause nephronophthisis type 2, linking renal cystic disease to the function of primary cilia and left-right axis determination

    PubMed Central

    Otto, Edgar A; Schermer, Bernhard; Obara, Tomoko; O'Toole, John F; Hiller, Karl S; Mueller, Adelheid M; Ruf, Rainer G; Hoefele, Julia; Beekmann, Frank; Landau, Daniel; Foreman, John W; Goodship, Judith A; Strachan, Tom; Kispert, Andreas; Wolf, Matthias T; Gagnadoux, Marie F; Nivet, Hubert; Antignac, Corinne; Walz, Gerd; Drummond, Iain A; Benzing, Thomas; Hildebrandt, Friedhelm

    2013-01-01

    Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, leads to chronic renal failure in children. The genes mutated in NPHP1 and NPHP4 have been identified, and a gene locus associated with infantile nephronophthisis (NPHP2) was mapped. The kidney phenotype of NPHP2 combines clinical features of NPHP and polycystic kidney disease (PKD). Here, we identify inversin (INVS) as the gene mutated in NPHP2 with and without situs inversus. We show molecular interaction of inversin with nephrocystin, the product of the gene mutated in NPHP1 and interaction of nephrocystin with β-tubulin, a main component of primary cilia. We show that nephrocystin, inversin and β-tubulin colocalize to primary cilia of renal tubular cells. Furthermore, we produce a PKD-like renal cystic phenotype and randomization of heart looping by knockdown of invs expression in zebrafish. The interaction and colocalization in cilia of inversin, nephrocystin and β-tubulin connect pathogenetic aspects of NPHP to PKD, to primary cilia function and to left-right axis determination. PMID:12872123

  9. Cilia and Diseases

    PubMed Central

    Brown, Jason M.; Witman, George B.

    2014-01-01

    In recent decades, cilia have moved from relative obscurity to a position of importance for understanding multiple complex human diseases. Now termed the ciliopathies, these diseases inflict devastating effects on millions of people worldwide. In this review, written primarily for teachers and students who may not yet be aware of the recent exciting developments in this field, we provide a general overview of our current understanding of cilia and human disease. We start with an introduction to cilia structure and assembly and indicate where they are found in the human body. We then discuss the clinical features of selected ciliopathies, with an emphasis on primary ciliary dyskinesia, polycystic kidney disease, and retinal degeneration. The history of ciliopathy research involves a fascinating interplay between basic and clinical sciences, highlighted in a timeline. Finally, we summarize the relative strengths of individual model organisms for ciliopathy research; many of these are suitable for classroom use. PMID:25960570

  10. 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

  11. Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia

    SciTech Connect

    Jumat, Muhammad Raihan; Yan, Yan; Ravi, Laxmi Iyer; Wong, Puisan; Huong, Tra Nguyen; Li, Chunwei; Tan, Boon Huan; Wang, De Yun; Sugrue, Richard J.

    2015-10-15

    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 the 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.

  12. Motile Cilia of Human Airway Epithelia Are Chemosensory

    PubMed Central

    Shah, Alok S; Ben-Shahar, Yehuda; Moninger, Thomas O; Kline, Joel N; Welsh, Michael J

    2010-01-01

    Cilia are microscopic projections that extend from eukaryotic cells. There are two general types of cilia; primary cilia serve as sensory organelles, whereas motile cilia exert mechanical force. The motile cilia emerging from human airway epithelial cells propel harmful inhaled material out of the lung. We found that these cells express sensory bitter taste receptors, which localized on motile cilia. Bitter compounds increased the intracellular Ca2+ concentration and stimulated ciliary beat frequency. Thus, airway epithelia contain a cell-autonomous system in which motile cilia both sense noxious substances entering airways and initiate a defensive mechanical mechanism to eliminate the offending compound. Hence, like primary cilia, classical motile cilia also contain sensors to detect the external environment. PMID:19628819

  13. Bone cell mechanosensation of fluid flow stimulation: a fluid-structure interaction model characterising the role integrin attachments and primary cilia.

    PubMed

    Vaughan, T J; Mullen, C A; Verbruggen, S W; McNamara, L M

    2015-08-01

    Load-induced fluid flow acts as an important biophysical signal for bone cell mechanotransduction in vivo, where the mechanical environment is thought to be monitored by integrin and primary cilia mechanoreceptors on the cell body. However, precisely how integrin- and primary cilia-based mechanosensors interact with the surrounding fluid flow stimulus and ultimately contribute to the biochemical response of bone cells within either the in vitro or in vivo environment remains poorly understood. In this study, we developed fluid-structure interaction models to characterise the deformation of integrin- and primary cilia-based mechanosensors in bone cells under fluid flow stimulation. Under in vitro fluid flow stimulation, these models predicted that integrin attachments on the cell-substrate interface were highly stimulated ε(eq) > 200,000 με, while the presence of a primary cilium on the cell also resulted in significant strain amplifications, arising at the ciliary base. As such, these mechanosensors likely play a role in mediating bone mechanotransduction in vitro. Under in vivo fluid flow stimulation, integrin attachments along the canalicular wall were highly stimulated and likely play a role in mediating cellular responses in vivo. The role of the primary cilium as a flow sensor in vivo depended upon its configuration within the lacunar cavity. Specifically, our results showed that a short free-standing primary cilium could not effectively fulfil a flow sensing role in vivo. However, a primary cilium that discretely attaches the lacunar wall can be highly stimulated, due to hydrodynamic pressure in the lacunocanalicular system and, as such, could play a role in mediating bone mechanotransduction in vivo. PMID:25399300

  14. The role of primary cilia in the development and disease of the retina

    PubMed Central

    Wheway, Gabrielle; Parry, David A; Johnson, Colin A

    2014-01-01

    The normal development and function of photoreceptors is essential for eye health and visual acuity in vertebrates. Mutations in genes encoding proteins involved in photoreceptor development and function are associated with a suite of inherited retinal dystrophies, often as part of complex multi-organ syndromic conditions. In this review, we focus on the role of the photoreceptor outer segment, a highly modified and specialized primary cilium, in retinal health and disease. We discuss the many defects in the structure and function of the photoreceptor primary cilium that can cause a class of inherited conditions known as ciliopathies, often characterized by retinal dystrophy and degeneration, and highlight the recent insights into disease mechanisms. PMID:24162842

  15. Dopamine receptors reveal an essential role of IFT-B, KIF17, and Rab23 in delivering specific receptors to primary cilia

    PubMed Central

    Leaf, Alison; Von Zastrow, Mark

    2015-01-01

    Appropriate physiological signaling by primary cilia depends on the specific targeting of particular receptors to the ciliary membrane, but how this occurs remains poorly understood. In this study, we show that D1-type dopaminergic receptors are delivered to cilia from the extra-ciliary plasma membrane by a mechanism requiring the receptor cytoplasmic tail, the intraflagellar transport complex-B (IFT-B), and ciliary kinesin KIF17. This targeting mechanism critically depends on Rab23, a small guanine nucleotide binding protein that has important effects on physiological signaling from cilia but was not known previously to be essential for ciliary delivery of any cargo. Depleting Rab23 prevents dopamine receptors from accessing the ciliary membrane. Conversely, fusion of Rab23 to a non-ciliary receptor is sufficient to drive robust, nucleotide-dependent mis-localization to the ciliary membrane. Dopamine receptors thus reveal a previously unrecognized mechanism of ciliary receptor targeting and functional role of Rab23 in promoting this process. DOI: http://dx.doi.org/10.7554/eLife.06996.001 PMID:26182404

  16. Multiple cilia suppress tumour formation.

    PubMed

    Eberhart, Charles

    2016-04-01

    Primary cilia are cellular structures that have important functions in development and disease. The suppression of multiciliate differentiation of choroid plexus precursors, and maintenance of a single primary cilium by Notch1, is now shown to be involved in choroid plexus tumour formation. PMID:27027488

  17. Cilia and coordination of signaling networks during heart development

    PubMed Central

    Koefoed, Karen; Veland, Iben Rønn; Pedersen, Lotte Bang; Larsen, Lars Allan; Christensen, Søren Tvorup

    2014-01-01

    Primary cilia are unique sensory organelles that coordinate a wide variety of different signaling pathways to control cellular processes during development and in tissue homeostasis. Defects in function or assembly of these antenna-like structures are therefore associated with a broad range of developmental disorders and diseases called ciliopathies. Recent studies have indicated a major role of different populations of cilia, including nodal and cardiac primary cilia, in coordinating heart development, and defects in these cilia are associated with congenital heart disease. Here, we present an overview of the role of nodal and cardiac primary cilia in heart development. PMID:24345806

  18. Cilia and cilia-associated proteins in cancer

    PubMed Central

    Seeger-Nukpezah, Tamina; Little, Joy L.; Serzhanova, Victoria; Golemis, Erica A.

    2013-01-01

    The primary cilium is a well-established target in the pathogenesis of numerous developmental and chronic disorders, and more recently is attracting interest as a structure relevant to cancer. Here we discuss mechanisms by which changes in cilia can contribute to the formation and growth of tumors. We emphasize the cancer-relevance of cilia-dependent signaling pathways and proteins including mTOR, VHL, TSC, WNT, Aurora-A, NEDD9, and Hedgehog, and highlight the emerging role of ciliary dysfunction in renal cell carcinoma, medulloblastoma, and breast cancer. PMID:24982684

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

    PubMed

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

    2016-02-01

    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

  20. The differential distribution of acetylated and detyrosinated alpha-tubulin in the microtubular cytoskeleton and primary cilia of hyaline cartilage chondrocytes

    PubMed Central

    POOLE, C. ANTHONY; ZHANG, ZI-JUN; ROSS, JACQUELINE M.

    2001-01-01

    The primary cilium is a ubiquitous cytoplasmic organelle of unknown function. Ultrastructural evidence of primary cilia in chondrocytes, and their colocalisation with the Golgi apparatus, has led to speculation that these structures are functionally linked. To investigate the relationship between these organelles, we examined the molecular anatomy of the microtubular cytoskeleton in the chondrocytes of chick embryo sterna. Thick cryosections were immunolabelled with antibodies directed against acetylated α-tubulin (C3B9), detyrosinated α-tubulin (ID5) and total α-tubulin (TAT), and imaged at high magnification using confocal laser scanning microscopy. Transmission electron microscopy confirmed the ultrastructure of the chondrocyte primary cilium and its structural relationship to the Golgi apparatus. Detyrosinated and acetylated α-tubulins were concentrated in the centrioles, centrosome and microtubule organising centre adjacent to the nucleus, with total α-tubulin distributed throughout the cytoplasm. ID5 stained the primary cilium at an incidence of 1 per cell, its colocalisation with C3B9 identifying the primary cilium as one of the most stable features of the microtubular cytoskeleton. Primary cilia varied from 1 to 4 μm in length, and 3 patterns of projection into the extracellular matrix were identified; (1) full extension and matrix contact, with minor undulations along the length; (2) partial extension and matrix contact, with a range of bending deflections; (3) cilium reclined against the cell surface with minimal matrix contact. Ultrastructural studies identified direct connections between extracellular collagen fibres and the proteins which decorate ciliary microtubules, suggesting a matrix–cilium–Golgi continuum in hyaline chondrocytes. These results strengthen the hypothesis that the primary cilium acts as a ‘cellular cybernetic probe' capable of transducing environmental information from the extracellular matrix, communicating this

  1. Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome

    PubMed Central

    Westlake, Christopher J.; Baye, Lisa M.; Nachury, Maxence V.; Wright, Kevin J.; Ervin, Karen E.; Phu, Lilian; Chalouni, Cecile; Beck, John S.; Kirkpatrick, Donald S.; Slusarski, Diane C.; Sheffield, Val C.; Scheller, Richard H.; Jackson, Peter K.

    2011-01-01

    Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly. We now describe serum-regulated upstream vesicular transport events leading to centrosomal Rab8 activation and ciliary membrane formation. Using live microscopy imaging, we show that upon serum withdrawal Rab8 is observed to assemble the ciliary membrane in ∼100 min. Rab8-dependent ciliary assembly is initiated by the relocalization of Rabin8 to Rab11-positive vesicles that are transported to the centrosome. After ciliogenesis, Rab8 ciliary transport is strongly reduced, and this reduction appears to be associated with decreased Rabin8 centrosomal accumulation. Rab11-GTP associates with the Rabin8 COOH-terminal region and is required for Rabin8 preciliary membrane trafficking to the centrosome and for ciliogenesis. Using zebrafish as a model organism, we show that Rabin8 and Rab11 are associated with the BBS pathway. Finally, using tandem affinity purification and mass spectrometry, we determined that the transport protein particle (TRAPP) II complex associates with the Rabin8 NH2-terminal domain and show that TRAPP II subunits colocalize with centrosomal Rabin8 and are required for Rabin8 preciliary targeting and ciliogenesis. PMID:21273506

  2. Sensory roles of neuronal cilia: cilia development, morphogenesis, and function in C. elegans.

    PubMed

    Bae, Young-Kyung; Barr, Maureen M

    2008-01-01

    In the free-living nematode Caenorhabditis elegans, cilia are found on the dendritic endings of sensory neurons. C. elegans cilia are classified as 'primary' or 'sensory' according to the '9+0' axonemal ultrastructure (nine doublet outer microtubules with no central microtubule pair) and lack of motility, characteristics of '9+2' cilia. The C. elegans ciliated nervous system allows the animal to perceive environmental stimuli and make appropriate developmental, physiological, and behavioral decisions. In vertebrates, the biological significance of primary cilia had been largely neglected. Recent findings have placed primary/sensory cilia in the center of cellular signaling and developmental processes. Studies using genetic model organisms such as C. elegans identified the link between ciliary dysfunction and human ciliopathies. Future studies in the worm will address important basic questions regarding ciliary development, morphogenesis, specialization, and signaling functions. PMID:18508635

  3. Polarisation of Light

    NASA Astrophysics Data System (ADS)

    Spottiswoode, William

    2015-01-01

    Preface; 1. Methods of polarisation; 2. Double refraction: polariscopes; 3. Chromatic polarisation: the wave theory; 4. Circular polarisation; 5. Circular polarisation by reflexion; 6. Phenomena produced by mechanical means: unannealed glass; 7. Atmospheric and other polarisation: the polar clock; 8. Rings and brushes produced by crystal plates; 9. Composition of colours by polarised light; Index.

  4. [Cilia and renal cysts].

    PubMed

    Paces-Fessy, Mélanie

    2014-11-01

    Advances in genomics, bioinformatics and the creation of model organisms have identified many genes associated with polycystic kidney diseases. Historically, these genes were not necessarily associated with ciliopathies, but it appeared that many connections can be made between the cystic kidney disease and function of the primary cilium. Indeed, the proteins encoded by these genes are localized to the cilium itself, to the basal body or are known to regulate the expression and localization of ciliary proteins. The goal of this article is to describe the multiple cellular processes that may lead to the development of renal cysts if they are deregulated. These include changes in proliferation rate, cell polarity or signaling pathways involved in embryonic kidney development. To highlight the role of the primary cilium in cystogenesis, I will discuss several studies investigating the function of ciliary genes and cilia in the kidneys of different model organisms. PMID:25388585

  5. Development and Distribution of Neuronal Cilia in Mouse Neocortex

    PubMed Central

    Arellano, Jon I.; Guadiana, Sarah M.; Breunig, Joshua J.; Rakic, Pasko; Sarkisian, Matthew R.

    2011-01-01

    Neuronal primary cilia are not generally recognized, but they are considered to extend from most, if not all, neurons in the neocortex. However, when and how cilia develop in neurons are not known. This study used immunohistochemistry for adenylyl cyclase III (ACIII), a marker of primary cilia, and electron microscopic analysis to describe the development and maturation of cilia in mouse neocortical neurons. Our results indicate that ciliogenesis is initiated in late fetal stages after neuroblast migration, when the mother centriole docks with the plasma membrane, becomes a basal body, and grows a cilia bud that we call a procilium. This procilium consists of a membranous protrusion extending from the basal body but lacking axonemal structure and remains undifferentiated until development of the axoneme and cilia elongation starts at about postnatal day 4. Neuronal cilia elongation and final cilia length depend on layer position, and the process extends for a long time, lasting 8–12 weeks. We show that, in addition to pyramidal neurons, inhibitory interneurons also grow cilia of comparable length, suggesting that cilia are indeed present in all neocortical neuron subtypes. Furthermore, the study of mice with defective ciliogenesis suggested that failed elongation of cilia is not essential for proper neuronal migration and laminar organization or establishment of neuronal polarity. Thus, the function of this organelle in neocortical neurons remains elusive. PMID:22020803

  6. Cilia, Wnt signaling, and the cytoskeleton.

    PubMed

    May-Simera, Helen L; Kelley, Matthew W

    2012-01-01

    Primary cilia have recently been highlighted as key regulators in development and disease. This review focuses on current work demonstrating the broad role of cilia-related proteins in developmental signaling systems. Of particular consideration is the importance of the basal body region, located at the base of the cilium, in its role as a focal point for many signaling pathways and as a microtubule organizing center. As the cilium is effectively a microtubular extension of the cytoskeleton, investigating connections between the cilium and the cytoskeleton provides greater insight into signaling and cell function. Of the many signaling pathways associated with primary cilia, the most extensively studied in association with the cytoskeleton and cytoskeletal rearrangements are both canonical and non-canonical Wnt pathways. One of the key concepts currently emerging is a possible additional role for the traditionally 'cilia-related' proteins in other aspects of cellular processes. In many cases, disruption of such processes manifests at the level of the cilium. While the involvement of cilia and cilia-related proteins in signaling pathways is currently being unraveled, there is a growing body of evidence to support the notion that ciliary proteins are required not only for regulation of Wnt signaling, but also as downstream effectors of Wnt signaling. This review summarizes recent advances in our understanding of the involvement of cilia and basal body proteins in Wnt signaling pathways. PMID:23351924

  7. KIF3A binds to β-arrestin for suppressing Wnt/β-catenin signalling independently of primary cilia in lung cancer

    PubMed Central

    Kim, Minsuh; Suh, Young-Ah; Oh, Ju-Hee; Lee, Bo Ra; Kim, Joon; Jang, Se Jin

    2016-01-01

    Aberrant Wnt/β-catenin signalling is implicated in the progression of several human cancers, including non-small cell lung cancer (NSCLC). However, mutations in Wnt/β-catenin pathway components are uncommon in NSCLC, and their epigenetic control remains unclear. Here, we show that KIF3A, a member of the kinesin-2 family, plays a role in suppressing Wnt/β-catenin signalling in NSCLC cells. KIF3A knockdown increases both β-catenin levels and transcriptional activity with concomitant promotion of malignant potential, such as increased proliferation and migration and upregulation of stemness markers. Because KIF3A binds β-arrestin, KIF3A depletion allows β-arrestin to form a complex with DVL2 and axin, stabilizing β-catenin. Although primary cilia, whose biogenesis requires KIF3A, are thought to restrain the Wnt response, pharmacological inhibition of ciliogenesis failed to increase β-catenin activity in NSCLC cells. A correlation between KIF3A loss and a poorer NSCLC prognosis as well as β-catenin and cyclin D1 upregulation further suggests that KIF3A suppresses Wnt/β-catenin signalling and tumourigenesis in NSCLC. PMID:27596264

  8. KIF3A binds to β-arrestin for suppressing Wnt/β-catenin signalling independently of primary cilia in lung cancer.

    PubMed

    Kim, Minsuh; Suh, Young-Ah; Oh, Ju-Hee; Lee, Bo Ra; Kim, Joon; Jang, Se Jin

    2016-01-01

    Aberrant Wnt/β-catenin signalling is implicated in the progression of several human cancers, including non-small cell lung cancer (NSCLC). However, mutations in Wnt/β-catenin pathway components are uncommon in NSCLC, and their epigenetic control remains unclear. Here, we show that KIF3A, a member of the kinesin-2 family, plays a role in suppressing Wnt/β-catenin signalling in NSCLC cells. KIF3A knockdown increases both β-catenin levels and transcriptional activity with concomitant promotion of malignant potential, such as increased proliferation and migration and upregulation of stemness markers. Because KIF3A binds β-arrestin, KIF3A depletion allows β-arrestin to form a complex with DVL2 and axin, stabilizing β-catenin. Although primary cilia, whose biogenesis requires KIF3A, are thought to restrain the Wnt response, pharmacological inhibition of ciliogenesis failed to increase β-catenin activity in NSCLC cells. A correlation between KIF3A loss and a poorer NSCLC prognosis as well as β-catenin and cyclin D1 upregulation further suggests that KIF3A suppresses Wnt/β-catenin signalling and tumourigenesis in NSCLC. PMID:27596264

  9. Mutations in CEP78 Cause Cone-Rod Dystrophy and Hearing Loss Associated with Primary-Cilia Defects.

    PubMed

    Nikopoulos, Konstantinos; Farinelli, Pietro; Giangreco, Basilio; Tsika, Chrysanthi; Royer-Bertrand, Beryl; Mbefo, Martial K; Bedoni, Nicola; Kjellström, Ulrika; El Zaoui, Ikram; Di Gioia, Silvio Alessandro; Balzano, Sara; Cisarova, Katarina; Messina, Andrea; Decembrini, Sarah; Plainis, Sotiris; Blazaki, Styliani V; Khan, Muhammad Imran; Micheal, Shazia; Boldt, Karsten; Ueffing, Marius; Moulin, Alexandre P; Cremers, Frans P M; Roepman, Ronald; Arsenijevic, Yvan; Tsilimbaris, Miltiadis K; Andréasson, Sten; Rivolta, Carlo

    2016-09-01

    Cone-rod degeneration (CRD) belongs to the disease spectrum of retinal degenerations, a group of hereditary disorders characterized by an extreme clinical and genetic heterogeneity. It mainly differentiates from other retinal dystrophies, and in particular from the more frequent disease retinitis pigmentosa, because cone photoreceptors degenerate at a higher rate than rod photoreceptors, causing severe deficiency of central vision. After exome analysis of a cohort of individuals with CRD, we identified biallelic mutations in the orphan gene CEP78 in three subjects from two families: one from Greece and another from Sweden. The Greek subject, from the island of Crete, was homozygous for the c.499+1G>T (IVS3+1G>T) mutation in intron 3. The Swedish subjects, two siblings, were compound heterozygotes for the nearby mutation c.499+5G>A (IVS3+5G>A) and for the frameshift-causing variant c.633delC (p.Trp212Glyfs(∗)18). In addition to CRD, these three individuals had hearing loss or hearing deficit. Immunostaining highlighted the presence of CEP78 in the inner segments of retinal photoreceptors, predominantly of cones, and at the base of the primary cilium of fibroblasts. Interaction studies also showed that CEP78 binds to FAM161A, another ciliary protein associated with retinal degeneration. Finally, analysis of skin fibroblasts derived from affected individuals revealed abnormal ciliary morphology, as compared to that of control cells. Altogether, our data strongly suggest that mutations in CEP78 cause a previously undescribed clinical entity of a ciliary nature characterized by blindness and deafness but clearly distinct from Usher syndrome, a condition for which visual impairment is due to retinitis pigmentosa. PMID:27588451

  10. Cilia and Polycystic Kidney Disease, Kith and Kin

    PubMed Central

    Huang, Liwei; Lipschutz, Joshua H.

    2015-01-01

    In the past decade, cilia have been found to play important roles in renal cystogenesis. Many genes, such as PKD1 and PKD2 which, when mutated, cause autosomal dominant polycystic kidney disease (ADPKD), have been found to localize to primary cilia. The cilium functions as a sensor to transmit extracellular signals into the cell. Abnormal cilia structure and function are associated with the development of polyscystic kidney disease (PKD). Cilia assembly includes centriole migration to the apical surface of the cell, ciliary vesicle docking and fusion with the cell membrane at the intended site of cilium outgrowth, and microtubule growth from the basal body. This review summarizes the most recent advances in cilia and PKD research, with special emphasis on the mechanisms of cytoplasmic and intraciliary protein transport during ciliogenesis. PMID:24898006

  11. Genetic Ablation of Type III Adenylyl Cyclase Exerts Region-Specific Effects on Cilia Architecture in the Mouse Nose

    PubMed Central

    Challis, Rosemary C.; Tian, Huikai; Yin, Wenbin; Ma, Minghong

    2016-01-01

    We recently reported that olfactory sensory neurons in the dorsal zone of the mouse olfactory epithelium exhibit drastic location-dependent differences in cilia length. Furthermore, genetic ablation of type III adenylyl cyclase (ACIII), a key olfactory signaling protein and ubiquitous marker for primary cilia, disrupts the cilia length pattern and results in considerably shorter cilia, independent of odor-induced activity. Given the significant impact of ACIII on cilia length in the dorsal zone, we sought to further investigate the relationship between cilia length and ACIII level in various regions throughout the mouse olfactory epithelium. We employed whole-mount immunohistochemical staining to examine olfactory cilia morphology in phosphodiesterase (PDE) 1C-/-;PDE4A-/- (simplified as PDEs-/- hereafter) and ACIII-/- mice in which ACIII levels are reduced and ablated, respectively. As expected, PDEs-/- animals exhibit dramatically shorter cilia in the dorsal zone (i.e., where the cilia pattern is found), similar to our previous observation in ACIII-/- mice. Remarkably, in a region not included in our previous study, ACIII-/- animals (but not PDEs-/- mice) have dramatically elongated, comet-shaped cilia, as opposed to characteristic star-shaped olfactory cilia. Here, we reveal that genetic ablation of ACIII has drastic, location-dependent effects on cilia architecture in the mouse nose. These results add a new dimension to our current understanding of olfactory cilia structure and regional organization of the olfactory epithelium. Together, these findings have significant implications for both cilia and sensory biology. PMID:26942602

  12. In vivo investigation of cilia structure and function using Xenopus

    PubMed Central

    Brooks, Eric R.; Wallingford, John B.

    2015-01-01

    Cilia are key organelles in development and homeostasis. The ever-expanding complement of cilia associated proteins necessitates rapid and tractable models for in vivo functional investigation. Xenopus laevis provides an attractive model for such studies, having multiple ciliated populations, including primary and multiciliated tissues. The rapid external development of Xenopus and the large cells make it an especially excellent platform for imaging studies. Here we present embryological and cell-biological methods for the investigation of cilia structure and function in Xenopus laevis, with a focus on quantitative live and fixed imaging. PMID:25837389

  13. Autophagy and regulation of cilia function and assembly

    PubMed Central

    Orhon, I; Dupont, N; Pampliega, O; Cuervo, A M; Codogno, P

    2015-01-01

    Motile and primary cilia (PC) are microtubule-based structures located at the cell surface of many cell types. Cilia govern cellular functions ranging from motility to integration of mechanical and chemical signaling from the environment. Recent studies highlight the interplay between cilia and autophagy, a conserved cellular process responsible for intracellular degradation. Signaling from the PC recruits the autophagic machinery to trigger autophagosome formation. Conversely, autophagy regulates ciliogenesis by controlling the levels of ciliary proteins. The cross talk between autophagy and ciliated structures is a novel aspect of cell biology with major implications in development, physiology and human pathologies related to defects in cilium function. PMID:25361082

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

    PubMed Central

    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. PMID:25553465

  15. 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

    SciTech Connect

    Chakravarthy, Balu; Gaudet, Chantal; Menard, Michel; Brown, Leslie; Atkinson, Trevor; LaFerla, Frank M.; Ito, Shingo; Armato, Ubaldo; Dal Pra, Ilaria; Whitfield, James

    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 somatostatin 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.

  16. Cyclic GMP and Cilia Motility

    PubMed Central

    Wyatt, Todd A.

    2015-01-01

    Motile cilia of the lungs respond to environmental challenges by increasing their ciliary beat frequency in order to enhance mucociliary clearance as a fundamental tenant of innate defense. One important second messenger in transducing the regulable nature of motile cilia is cyclic guanosine 3′,5′-monophosphate (cGMP). In this review, the history of cGMP action is presented and a survey of the existing data addressing cGMP action in ciliary motility is presented. Nitric oxide (NO)-mediated regulation of cGMP in ciliated cells is presented in the context of alcohol-induced cilia function and dysfunction. PMID:26264028

  17. Olfactory Cilia: Linking Sensory Cilia Function and Human Disease

    PubMed Central

    Jenkins, Paul M.; McEwen, Dyke P.

    2009-01-01

    The olfactory system gives us an awareness of our immediate environment by allowing us to detect airborne stimuli. The components necessary for detection of these odorants are compartmentalized in the cilia of olfactory sensory neurons. Cilia are microtubule-based organelles, which can be found projecting from the surface of almost any mammalian cell, and are critical for proper olfactory function. Mislocalization of ciliary proteins and/or the loss of cilia cause impaired olfactory function, which is now recognized as a clinical manifestation of a broad class of human diseases, termed ciliopathies. Future work investigating the mechanisms of olfactory cilia function will provide us important new information regarding the pathogenesis of human sensory perception diseases. PMID:19406873

  18. Antennas of organ morphogenesis: the roles of cilia in vertebrate kidney development.

    PubMed

    Marra, Amanda N; Li, Yue; Wingert, Rebecca A

    2016-09-01

    Cilia arose early during eukaryotic evolution, and their structural components are highly conserved from the simplest protists to complex metazoan species. In recent years, the role of cilia in the ontogeny of vertebrate organs has received increasing attention due to a staggering correlation between human disease and dysfunctional cilia. In particular, the presence of cilia in both the developing and mature kidney has become a deep area of research due to ciliopathies common to the kidney, such as polycystic kidney disease (PKD). Interestingly, mutations in genes encoding proteins that localize to the cilia cause similar cystic phenotypes in kidneys of various vertebrates, suggesting an essential role for cilia in kidney organogenesis and homeostasis as well. Importantly, the genes so far identified in kidney disease have conserved functions across species, whose kidneys include both primary and motile cilia. Here, we aim to provide a comprehensive description of cilia and their role in kidney development, as well as highlight the usefulness of the zebrafish embryonic kidney as a model to further understand the function of cilia in kidney health. PMID:27389733

  19. Evaluating efficiency and robustness in cilia design

    NASA Astrophysics Data System (ADS)

    Guo, Hanliang; Kanso, Eva

    2016-03-01

    Motile cilia are used by many eukaryotic cells to transport flow. Cilia-driven flows are important to many physiological functions, yet a deep understanding of the interplay between the mechanical structure of cilia and their physiological functions in healthy and diseased conditions remains elusive. To develop such an understanding, one needs a quantitative framework to assess cilia performance and robustness when subject to perturbations in the cilia apparatus. Here we link cilia design (beating patterns) to function (flow transport) in the context of experimentally and theoretically derived cilia models. We particularly examine the optimality and robustness of cilia design. Optimality refers to efficiency of flow transport, while robustness is defined as low sensitivity to variations in the design parameters. We find that suboptimal designs can be more robust than optimal ones. That is, designing for the most efficient cilium does not guarantee robustness. These findings have significant implications on the understanding of cilia design in artificial and biological systems.

  20. Evaluating efficiency and robustness in cilia design.

    PubMed

    Guo, Hanliang; Kanso, Eva

    2016-03-01

    Motile cilia are used by many eukaryotic cells to transport flow. Cilia-driven flows are important to many physiological functions, yet a deep understanding of the interplay between the mechanical structure of cilia and their physiological functions in healthy and diseased conditions remains elusive. To develop such an understanding, one needs a quantitative framework to assess cilia performance and robustness when subject to perturbations in the cilia apparatus. Here we link cilia design (beating patterns) to function (flow transport) in the context of experimentally and theoretically derived cilia models. We particularly examine the optimality and robustness of cilia design. Optimality refers to efficiency of flow transport, while robustness is defined as low sensitivity to variations in the design parameters. We find that suboptimal designs can be more robust than optimal ones. That is, designing for the most efficient cilium does not guarantee robustness. These findings have significant implications on the understanding of cilia design in artificial and biological systems. PMID:27078459

  1. Type 3 Adenylyl Cyclase and Somatostatin Receptor 3 Expression Persists in Aged Rat Neocortical and Hippocampal Neuronal Cilia

    PubMed Central

    Guadiana, Sarah M.; Parker, Alexander K.; Filho, Gileno F.; Sequeira, Ashton; Semple-Rowland, Susan; Shaw, Gerry; Mandel, Ronald J.; Foster, Thomas C.; Kumar, Ashok; Sarkisian, Matthew R.

    2016-01-01

    The primary cilia of forebrain neurons assemble around birth and become enriched with neuromodulatory receptors. Our understanding of the permanence of these structures and their associated signaling pathways in the aging brain is poor, but they are worthy of investigation because disruptions in neuronal cilia signaling have been implicated in changes in learning and memory, depression-like symptoms, and sleep anomalies. Here, we asked whether neurons in aged forebrain retain primary cilia and whether the staining characteristics of aged cilia for type 3 adenylyl cyclase (ACIII), somatostatin receptor 3 (SSTR3), and pericentrin resemble those of cilia in younger forebrain. To test this, we analyzed immunostained sections of forebrain tissues taken from young and aged male Fischer 344 (F344) and F344 × Brown Norway (F344 × BN) rats. Analyses of ACIII and SSTR3 in young and aged cortices of both strains of rats revealed that the staining patterns in the neocortex and hippocampus were comparable. Virtually every NeuN positive cell examined possessed an ACIII positive cilium. The lengths of ACIII positive cilia in neocortex were similar between young and aged for both strains, whereas in F344 × BN hippocampus, the cilia lengths increased with age in CA1 and CA3, but not in dentate gyrus (DG). Additionally, the percentages of ACIII positive cilia that were also SSTR3 positive did not differ between young and aged tissues in either strain. We also found that pericentrin, a protein that localizes to the basal bodies of neuronal cilia and functions in primary cilia assembly, persisted in aged cortical neurons of both rat strains. Collectively, our data show that neurons in aged rat forebrain possess primary cilia and that these cilia, like those present in younger brain, continue to localize ACIII, SSTR3, and pericentrin. Further studies will be required to determine if the function and signaling pathways regulated by cilia are similar in aged compared to young brain

  2. Polarised light sheet tomography.

    PubMed

    Reidt, Sascha L; O'Brien, Daniel J; Wood, Kenneth; MacDonald, Michael P

    2016-05-16

    The various benefits of light sheet microscopy have made it a widely used modality for capturing three-dimensional images. It is mostly used for fluorescence imaging, but recently another technique called light sheet tomography solely relying on scattering was presented. The method was successfully applied to imaging of plant roots in transparent soil, but is limited when it comes to more turbid samples. This study presents a polarised light sheet tomography system and its advantages when imaging in highly scattering turbid media. The experimental configuration is guided by Monte Carlo radiation transfer methods, which model the propagation of a polarised light sheet in the sample. Images of both reflecting and absorbing phantoms in a complex collagenous matrix were acquired, and the results for different polarisation configurations are compared. Focus scanning methods were then used to reduce noise and produce three-dimensional reconstructions of absorbing targets. PMID:27409945

  3. Polarisation properties of pulsars at optical wavelengths

    NASA Astrophysics Data System (ADS)

    Mignani, Roberto; Marelli, Martino; Shearer, Andrew; Slowikowska, Agnieszka

    2016-07-01

    Polarisation measurements of pulsars offer unique insights into their highly-magnetised relativistic environments and represent a primary test for neutron star magnetosphere models and radiation emission mechanisms. Besides the radio band, optical observations have been, so far, best suited to these goals, with polarisation measurements in the X-rays becoming possible in the near future thanks to missions, such as XIPE and IXPE. In this talk, we review the status of the optical polarisation measurements of pulsars and we foresee possible synergies between X-ray polarimetry observations of selected pulsars with, e.g XIPE and IXPE, and optical observations with the next generation of extremely large telescope, such as the E-ELT.

  4. Tau polarisation at LEP

    NASA Astrophysics Data System (ADS)

    Alemany, Ricard

    1999-04-01

    The measurements of the tau polarisation at LEP I are reviewed. Special emphasis is given to the new preliminary results presented at this conference. The ALEPH collaboration has studied the polarisation as a function of the polar angle using a new method based on the tau direction reconstruction and fully exploiting the angular correlations. A second traditional approach, based on the single tau decays has been also developed. The DELPHI collaboration has also studied the full data sample using an individual tau decay method and an inclusive hadronic selection. The results from the four experiments are presented with discussion of the compatibility among the methods and experiments.

  5. 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

  6. Emergence of metachronal waves in cilia arrays

    PubMed Central

    Elgeti, Jens; Gompper, Gerhard

    2013-01-01

    Propulsion by cilia is a fascinating and universal mechanism in biological organisms to generate fluid motion on the cellular level. Cilia are hair-like organelles, which are found in many different tissues and many uni- and multicellular organisms. Assembled in large fields, cilia beat neither randomly nor completely synchronously—instead they display a striking self-organization in the form of metachronal waves (MCWs). It was speculated early on that hydrodynamic interactions provide the physical mechanism for the synchronization of cilia motion. Theory and simulations of physical model systems, ranging from arrays of highly simplified actuated particles to a few cilia or cilia chains, support this hypothesis. The main questions are how the individual cilia interact with the flow field generated by their neighbors and synchronize their beats for the metachronal wave to emerge and how the properties of the metachronal wave are determined by the geometrical arrangement of the cilia, like cilia spacing and beat direction. Here, we address these issues by large-scale computer simulations of a mesoscopic model of 2D cilia arrays in a 3D fluid medium. We show that hydrodynamic interactions are indeed sufficient to explain the self-organization of MCWs and study beat patterns, stability, energy expenditure, and transport properties. We find that the MCW can increase propulsion velocity more than 3-fold and efficiency almost 10-fold—compared with cilia all beating in phase. This can be a vital advantage for ciliated organisms and may be interesting to guide biological experiments as well as the design of efficient microfluidic devices and artificial microswimmers. PMID:23487771

  7. Emergence of metachronal waves in cilia arrays.

    PubMed

    Elgeti, Jens; Gompper, Gerhard

    2013-03-19

    Propulsion by cilia is a fascinating and universal mechanism in biological organisms to generate fluid motion on the cellular level. Cilia are hair-like organelles, which are found in many different tissues and many uni- and multicellular organisms. Assembled in large fields, cilia beat neither randomly nor completely synchronously--instead they display a striking self-organization in the form of metachronal waves (MCWs). It was speculated early on that hydrodynamic interactions provide the physical mechanism for the synchronization of cilia motion. Theory and simulations of physical model systems, ranging from arrays of highly simplified actuated particles to a few cilia or cilia chains, support this hypothesis. The main questions are how the individual cilia interact with the flow field generated by their neighbors and synchronize their beats for the metachronal wave to emerge and how the properties of the metachronal wave are determined by the geometrical arrangement of the cilia, like cilia spacing and beat direction. Here, we address these issues by large-scale computer simulations of a mesoscopic model of 2D cilia arrays in a 3D fluid medium. We show that hydrodynamic interactions are indeed sufficient to explain the self-organization of MCWs and study beat patterns, stability, energy expenditure, and transport properties. We find that the MCW can increase propulsion velocity more than 3-fold and efficiency almost 10-fold--compared with cilia all beating in phase. This can be a vital advantage for ciliated organisms and may be interesting to guide biological experiments as well as the design of efficient microfluidic devices and artificial microswimmers. PMID:23487771

  8. 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.

  9. 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

  10. Swimming like algae: biomimetic soft artificial cilia.

    PubMed

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

    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

  11. 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

  12. Microfluidic manipulation with artificial/bioinspired cilia.

    PubMed

    den Toonder, Jaap M J; Onck, Patrick R

    2013-02-01

    A recent development, inspired by nature, is the use of 'artificial cilia' to create pumping and/or mixing in microfluidic devices. Cilia are small hairs that can be found in biology and are used for (fluid) actuation and sensing. Microscopic actuators resembling cilia, actuated to move under the influence of various stimuli such as electrostatic field, magnetic field, and even light, have been developed by a number of groups and shown to be capable of generating flow and mixing in microfluidic environments. The research on artificial cilia started about a decade ago and is rapidly expanding. In addition to being relevant for potential application in lab-on-a-chip devices, the work on artificial cilia forms a beautiful example of how a biological system can form the successful basis for both scientific research and technological applications. In this review, we will give an overview of the most important approaches in this exciting field. PMID:23245658

  13. Loss of Bardet–Biedl syndrome proteins alters the morphology and function of motile cilia in airway epithelia

    PubMed Central

    Shah, Alok S.; Farmen, Sara L.; Moninger, Thomas O.; Businga, Thomas R.; Andrews, Michael P.; Bugge, Kevin; Searby, Charles C.; Nishimura, Darryl; Brogden, Kim A.; Kline, Joel N.; Sheffield, Val C.; Welsh, Michael J.

    2008-01-01

    Mutations in a group of genes that contribute to ciliary function cause Bardet–Biedl syndrome (BBS). Most studies of BBS have focused on primary, sensory cilia. Here, we asked whether loss of BBS proteins would also affect motile cilia lining the respiratory tract. We found that BBS genes were expressed in human airway epithelia, and BBS2 and BBS4 localized to cellular structures associated with motile cilia. Although BBS proteins were not required for ciliogenesis, their loss caused structural defects in a fraction of cilia covering mouse airway epithelia. The most common abnormality was bulges filled with vesicles near the tips of cilia. We discovered this same misshapen appearance in airway cilia from Bbs1, Bbs2, Bbs4, and Bbs6 mutant mice. The structural abnormalities were accompanied by functional defects; ciliary beat frequency was reduced in Bbs mutant mice. Previous reports suggested BBS might increase the incidence of asthma. However, compared with wild-type controls, neither airway hyperresponsiveness nor inflammation increased in Bbs2−/− or Bbs4−/− mice immunized with ovalbumin. Instead, these animals were partially protected from airway hyperresponsiveness. These results emphasize the role of BBS proteins in both the structure and function of motile cilia. They also invite additional scrutiny of motile cilia dysfunction in patients with this disease. PMID:18299575

  14. Polarised neutron scattering from dynamic polarised targets in biology

    NASA Astrophysics Data System (ADS)

    Knop, W.; Hirai, M.; Olah, G.; Meerwinck, W.; Schink, H.-J.; Stuhrman, H. B.; Wagner, R.; Wenkow-EsSouni, M.; Zhao, J.; Schärpf, O.; Crichton, R. R.; Krumpolc, M.; Nierhaus, K. H.; Niinikoski, T. O.; Rijllart, A.

    1991-10-01

    The contrast giving rise to neutron small-angle scattering can be enhanced considerably by polarisation of the hydrogen nuclei [J. des Coizeaux and G. Jannink, Les Polymères en Solution, Les Editions de Physique, F-91944 Les Ulis, France (1987)]. Using polarised neutrons the scattering from protonated labels in a deuterated matrix will increase by an order of magnitude. This is the basis of nuclear spin contrast variation, a method which is of particular interest for the in situ structure determination of macromolecular components. A new polarised target for neutron scattering has been designed by CERN and tested successfully at FRG-1 of the GKSS research centre. For the purpose of thermal-neutron scattering the frozen solutions of biomolecules are immersed in liquid helium 4, which is thermally coupled to the cooling mixture of helium 3/helium 4 of the dilution refrigerator. The nuclear spins are aligned with respect to the external magnetic field-parallel or antiparallel-by dynamic nuclear polarisation (DNP). The gain in neutron scattering compared to earlier experiments using direct cooling of the sample by helium 3 is a factor of 30. Another factor of 30 arises from the installation of the cold source and the beryllium reflector in FRG-1 [W. Knop et al., J. Appl. Cryst. 22 (1989) 352]. Pure nuclear spin targets are produced from dynamic polarised targets by selective depolarisation. In biological material only the hydrogen isotopes contribute significantly to polarised neutron scattering. Thus, saturation of the proton NMR yields a deuteron target, provided the target material has been enriched by the latter isotope. A proton target is obtained from the dynamic polarised target by saturation of deuteron NMR. This leads to six additional scattering functions reflecting the proton and deuteron spin densities and the correlations between the polarised isotopes. Polarised neutron scattering from nuclear spin targets of apoferritin and various derivatives of the

  15. Centrosomal protein CP110 controls maturation of the mother centriole during cilia biogenesis

    PubMed Central

    Yadav, Sharda Prasad; Sharma, Neel Kamal; Liu, Chunqiao; Dong, Lijin; Li, Tiansen; Swaroop, Anand

    2016-01-01

    ABSTRACT Defects in cilia centrosomal genes cause pleiotropic clinical phenotypes, collectively called ciliopathies. Cilia biogenesis is initiated by the interaction of positive and negative regulators. Centriolar coiled coil protein 110 (CP110) caps the distal end of the mother centriole and is known to act as a suppressor to control the timing of ciliogenesis. Here, we demonstrate that CP110 promotes cilia formation in vivo, in contrast to findings in cultured cells. Cp110−/− mice die shortly after birth owing to organogenesis defects as in ciliopathies. Shh signaling is impaired in null embryos and primary cilia are reduced in multiple tissues. We show that CP110 is required for anchoring of basal bodies to the membrane during cilia formation. CP110 loss resulted in an abnormal distribution of core components of subdistal appendages (SDAs) and of recycling endosomes, which may be associated with premature extension of axonemal microtubules. Our data implicate CP110 in SDA assembly and ciliary vesicle docking, two requisite early steps in cilia formation. We suggest that CP110 has unique context-dependent functions, acting as both a suppressor and a promoter of ciliogenesis. PMID:26965371

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

    PubMed

    Breslow, David K; Koslover, Elena F; Seydel, Federica; Spakowitz, Andrew J; Nachury, Maxence V

    2013-10-14

    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

  17. 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

  18. Manipulating Cilia Using the 3DFM

    NASA Astrophysics Data System (ADS)

    Fisher, Jay K.; O'Brien, E. Timothy; Taylor, R. M.; Davis, C. W.; Matsui, H.; Vicci, L.; Matthews, G.; Cribb, J.; Desai, K.; Wilde, B.; Superfine, R.

    2003-11-01

    Mucus flow generated by beating cilia projecting from epithelial cells is responsible for the removal of pathogens in the lungs. When the hydrodynamics of this mucociliary clearance system fails, as happens in the condition of Cystic Fibrosis, ensuing infections can destroy the lungs. A complex phenomenology includes the force generation of the cilia, the coupling of the cilia tips to the overlying mucus during the power stroke, and finally, it is speculated that the cilia act as force sensors to allow the control of the mucus viscosity and volume. We have designed a system that allows the measurement of 3 dimensional forces within an optical microscope using magnetic forces and superparamagnetic beads. We have functionalized the beads with antibodies and attached them to the cilia of human lung cell cultures. A laser is focused onto the bead whose motion is tracked using back focal plane detection of the forward scattered light. We have measured the 3 dimensional trajectory of beads attached to cilia beating up to 15Hz in human lung cell cultures with 10 nm, 1msec resolution. When forces are applied to the bead, we observe changes in the trajectory and velocity, implying a strong mechanoresponse of the cell as sensed by the cilia.

  19. Reduced cilia frequencies in human renal cell carcinomas versus neighboring parenchymal tissue

    PubMed Central

    2013-01-01

    eighty-nine clear cell, eight papillary, five chromophobe renal cell carcinomas, two sarcomatoid renal tumors and six oncocytomas were determined. A marked decrease of primary cilia across renal cell carcinoma subtypes was observed compared to adjacent nontumorigenic tissue. Conclusions Our study shows that cilia are predominantly lost in renal cell carcinomas compared to tissue of the tumor parenchyma. These results suggest that ciliary loss is common in renal tumorigenesis, possibly participating in the sequence of cellular events leading to malignant tumor development. Future therapies aimed at restoring or circumventing cilia signaling might therefore aid in current treatment efficacy. PMID:23369289

  20. Specialized Cilia in Mammalian Sensory Systems

    PubMed Central

    Falk, Nathalie; Lösl, Marlene; Schröder, Nadja; Gießl, Andreas

    2015-01-01

    Cilia and flagella are highly conserved and important microtubule-based organelles that project from the surface of eukaryotic cells and act as antennae to sense extracellular signals. Moreover, cilia have emerged as key players in numerous physiological, developmental, and sensory processes such as hearing, olfaction, and photoreception. Genetic defects in ciliary proteins responsible for cilia formation, maintenance, or function underlie a wide array of human diseases like deafness, anosmia, and retinal degeneration in sensory systems. Impairment of more than one sensory organ results in numerous syndromic ciliary disorders like the autosomal recessive genetic diseases Bardet-Biedl and Usher syndrome. Here we describe the structure and distinct functional roles of cilia in sensory organs like the inner ear, the olfactory epithelium, and the retina of the mouse. The spectrum of ciliary function in fundamental cellular processes highlights the importance of elucidating ciliopathy-related proteins in order to find novel potential therapies. PMID:26378583

  1. 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.

  2. DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes.

    PubMed

    Dougherty, Gerard W; Loges, Niki T; Klinkenbusch, Judith A; Olbrich, Heike; Pennekamp, Petra; Menchen, Tabea; Raidt, Johanna; Wallmeier, Julia; Werner, Claudius; Westermann, Cordula; Ruckert, Christian; Mirra, Virginia; Hjeij, Rim; Memari, Yasin; Durbin, Richard; Kolb-Kokocinski, Anja; Praveen, Kavita; Kashef, Mohammad A; Kashef, Sara; Eghtedari, Fardin; Häffner, Karsten; Valmari, Pekka; Baktai, György; Aviram, Micha; Bentur, Lea; Amirav, Israel; Davis, Erica E; Katsanis, Nicholas; Brueckner, Martina; Shaposhnykov, Artem; Pigino, Gaia; Dworniczak, Bernd; Omran, Heymut

    2016-08-01

    Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography. PMID:26909801

  3. Developmental Signaling: Does It Bridge the Gap Between Cilia Dysfunction and Renal Cystogenesis?

    PubMed Central

    Tran, Pamela V.; Sharma, Madhulika; Li, Xiaogang; Calvet, James P.

    2015-01-01

    For more than a decade, evidence has accumulated linking dysfunction of primary cilia to renal cystogenesis, yet molecular mechanisms remain undefined. The pathogenesis of renal cysts is complex, involving multiple cellular aberrations and signaling pathways. Adding to this complexity, primary cilia exhibit multiple roles in a context-dependent manner. On renal epithelial cells, primary cilia act as mechanosensors and trigger extracellular Ca2+ influx in response to laminar fluid flow. During mammalian development, primary cilia mediate the Hedgehog (Hh), Wnt, and Notch pathways, which control cell proliferation and differentiation, and tissue morphogenesis. Further, experimental evidence suggests the developmental state of the kidney strongly influences renal cystic disease. Thus, we review evidence for regulation of Ca2+ and cAMP, key molecules in renal cystogenesis, at the primary cilium, the role of Hh, Wnt, and Notch signaling in renal cystic disease, and the interplay between these developmental pathways and Ca2+ signaling. Indeed if these developmental pathways influence renal cystogenesis, these may represent novel therapeutic targets that can be integrated into a combination therapy for renal cystic disease. PMID:24861210

  4. The zebrafish foxj1a transcription factor regulates cilia function in response to injury and epithelial stretch.

    PubMed

    Hellman, Nathan E; Liu, Yan; Merkel, Erin; Austin, Christina; Le Corre, Stephanie; Beier, David R; Sun, Zhaoxia; Sharma, Neeraj; Yoder, Bradley K; Drummond, Iain A

    2010-10-26

    Cilia are essential for normal organ function and developmental patterning, but their role in injury and regeneration responses is unknown. To probe the role of cilia in injury, we analyzed the function of foxj1, a transcriptional regulator of cilia genes, in response to tissue damage and renal cyst formation. Zebrafish foxj1a, but not foxj1b, was rapidly induced in response to epithelial distension and stretch, kidney cyst formation, acute kidney injury by gentamicin, and crush injury in spinal cord cells. Obstruction-induced up-regulation of foxj1a was not inhibited by cycloheximide, identifying foxj1a as a primary response gene to epithelial injury. Foxj1 was also dramatically up-regulated in murine cystic kidney disease epithelia [jck/jck (nek8) and Ift88Tg737Rpw(-/-)] as well as in response to kidney ischemia-reperfusion injury. Obstruction of the zebrafish pronephric tubule caused a rapid increase in cilia beat rate that correlated tightly with expanded tubule diameter and epithelial stretch. Zebrafish foxj1a was specifically required for cilia motility. Enhanced foxj1a expression in obstructed tubules induced cilia motility target genes efhc1, tektin-1, and dnahc9. foxj1a-deficient embryos failed to up-regulate efhc1, tektin-1, and dnahc9 and could not maintain enhanced cilia beat rates after obstruction, identifying an essential role for foxj1 in modulating cilia function after injury. These studies reveal that activation of a Foxj1 transcriptional network of ciliogenic genes is an evolutionarily conserved response to multiple forms of tissue damage and highlight enhanced cilia function as a previously uncharacterized component of organ homeostasis. PMID:20937855

  5. 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

  6. The essential roles of transition fibers in the context of cilia

    PubMed Central

    Wei, Qing; Ling, Kun; Hu, Jinghua

    2015-01-01

    Once thought of as a vestigial organelle, the primary cilium is now recognized as a signaling hub for key cellular pathways in vertebrate development. The recent renaissance in cilia studies significantly improved our understanding of how cilia form and function, but little is known about how ciliogenesis is initiated and how ciliary proteins enter cilia. These important ciliary events require transition fibers (TFs) that are positioned at the ciliary base as symmetric nine-bladed propeller fibrous structures. Up until recently, TFs have been the most underappreciated ciliary structures due to limited knowledge about their molecular composition and function. Here, we highlight recent advances in our understanding of TF composition and the indispensable roles of TFs in regulating the initiation of ciliogenesis and the selective import of ciliary proteins. PMID:25988548

  7. Mutations in the Cilia Gene ARL13B Lead to the Classical Form of Joubert Syndrome

    PubMed Central

    Cantagrel, Vincent; Silhavy, Jennifer L.; Bielas, Stephanie L.; Swistun, Dominika; Marsh, Sarah E.; Bertrand, Julien Y.; Audollent, Sophie; Attié-Bitach, Tania; Holden, Kenton R.; Dobyns, William B.; Traver, David; Al-Gazali, Lihadh; Ali, Bassam R.; Lindner, Tom H.; Caspary, Tamara; Otto, Edgar A.; Hildebrandt, Friedhelm; Glass, Ian A.; Logan, Clare V.; Johnson, Colin A.; Bennett, Christopher; Brancati, Francesco; Valente, Enza Maria; Woods, C. Geoffrey; Gleeson, Joseph G.

    2008-01-01

    Joubert syndrome (JS) and related disorders are a group of autosomal-recessive conditions sharing the “molar tooth sign” on axial brain MRI, together with cerebellar vermis hypoplasia, ataxia, and psychomotor delay. JS is suggested to be a disorder of cilia function and is part of a spectrum of disorders involving retinal, renal, digital, oral, hepatic, and cerebral organs. We identified mutations in ARL13B in two families with the classical form of JS. ARL13B belongs to the Ras GTPase family, and in other species is required for ciliogenesis, body axis formation, and renal function. The encoded Arl13b protein was expressed in developing murine cerebellum and localized to the cilia in primary neurons. Overexpression of human wild-type but not patient mutant ARL13B rescued the Arl13b scorpion zebrafish mutant. Thus, ARL13B has an evolutionarily conserved role mediating cilia function in multiple organs. PMID:18674751

  8. Stall Force and Response of Lung Cilia

    NASA Astrophysics Data System (ADS)

    Superfine, Richard; Hill, David; Swaminathan, Vinay; O'Brien, E. Timothy; Boucher, Ric; Button, Brian; Estes, Ashley

    2008-03-01

    We report on the response of lung cilia to applied forces. We have applied magnetic forces to magnetic beads attached to individual human lung cilia in cell cultures. Our magnetic system is capable of generating large forces (˜1nanoNewton on 1 micron beads) with a 3kHz bandwidth. We record the cilia beat motion using video microscopy to record beat frequency and amplitude as a function of applied force. We present three major findings. First, the stall force is approximately 150 pN. Second the frequency is unchanged by the application of forces up to the stall point. Third, the speed of the beat motion slows down according to the diminution of the beat amplitude while maintaining a constant frequency and the speed of the motion is the same whether the beat direction is in the same direction as the applied force or against the applied force.

  9. RFX3 governs growth and beating efficiency of motile cilia in mouse and controls the expression of genes involved in human ciliopathies.

    PubMed

    El Zein, Loubna; Ait-Lounis, Aouatef; Morlé, Laurette; Thomas, Joëlle; Chhin, Brigitte; Spassky, Nathalie; Reith, Walter; Durand, Bénédicte

    2009-09-01

    Cilia are cellular organelles that play essential physiological and developmental functions in various organisms. They can be classified into two categories, primary cilia and motile cilia, on the basis of their axonemal architecture. Regulatory factor X (RFX) transcription factors have been shown to be involved in the assembly of primary cilia in Caenorhabditis elegans, Drosophila and mice. Here, we have taken advantage of a novel primary-cell culture system derived from mouse brain to show that RFX3 is also necessary for biogenesis of motile cilia. We found that the growth and beating efficiencies of motile cilia are impaired in multiciliated Rfx3(-/-) cells. RFX3 was required for optimal expression of the FOXJ1 transcription factor, a key player in the differentiation program of motile cilia. Furthermore, we demonstrate for the first time that RFX3 regulates the expression of axonemal dyneins involved in ciliary motility by binding directly to the promoters of their genes. In conclusion, RFX proteins not only regulate genes involved in ciliary assembly, but also genes that are involved in ciliary motility and that are associated with ciliopathies such as primary ciliary dyskinesia in humans. PMID:19671664

  10. 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

  11. Cottingham formula and nucleon polarisabilities

    NASA Astrophysics Data System (ADS)

    Gasser, J.; Hoferichter, M.; Leutwyler, H.; Rusetsky, A.

    2015-08-01

    The difference between the electromagnetic self-energies of proton and neutron can be calculated with the Cottingham formula, which expresses the self-energies as an integral over the electroproduction cross sections - provided the nucleon matrix elements of the current commutator do not contain a fixed pole. We show that, under the same proviso, the subtraction function occurring in the dispersive representation of the virtual Compton forward scattering amplitude is determined by the cross sections. The representation in particular leads to a parameter-free sum rule for the nucleon polarisabilities. We evaluate the sum rule for the difference between the electric polarisabilities of proton and neutron by means of the available parameterisations of the data and compare the result with experiment.

  12. Regeneration of cilia in heavily irradiated sea urchin embryos

    SciTech Connect

    Rustad, R.C.

    1981-12-01

    Cilia were removed from blastulae, gastrulae, and plutei of the sea urchins Arbacia punctulata and Lytechinus variegatus by shaking the embryos in hypertonic media. Exposure to 50 krad (and in some experiments 100 krad) of ..gamma.. radiation either before or after deciliation had no effect on the time of appearance of regenerating cilia. There were no visually obvious differences in the rate of growth of the cilia in control and irradiated embryos. The cilia commenced beating at the same time, but the initial beating sometimes seemed less vigorous following irradiation. The data support the hypothesis that radiation has no major effect on the assembly from mature basal bodies of the microtubules of cilia.

  13. Cildb: a knowledgebase for centrosomes and cilia

    PubMed Central

    Arnaiz, Olivier; Malinowska, Agata; Klotz, Catherine; Sperling, Linda; Dadlez, Michal; Koll, France; Cohen, Jean

    2009-01-01

    Ciliopathies, pleiotropic diseases provoked by defects in the structure or function of cilia or flagella, reflect the multiple roles of cilia during development, in stem cells, in somatic organs and germ cells. High throughput studies have revealed several hundred proteins that are involved in the composition, function or biogenesis of cilia. The corresponding genes are potential candidates for orphan ciliopathies. To study ciliary genes, model organisms are used in which particular questions on motility, sensory or developmental functions can be approached by genetics. In the course of high throughput studies of cilia in Paramecium tetraurelia, we were confronted with the problem of comparing our results with those obtained in other model organisms. We therefore developed a novel knowledgebase, Cildb, that integrates ciliary data from heterogeneous sources. Cildb links orthology relationships among 18 species to high throughput ciliary studies, and to OMIM data on human hereditary diseases. The web interface of Cildb comprises three tools, BioMart for complex queries, BLAST for sequence homology searches and GBrowse for browsing the human genome in relation to OMIM information for human diseases. Cildb can be used for interspecies comparisons, building candidate ciliary proteomes in any species, or identifying candidate ciliopathy genes. Database URL: http://cildb.cgm.cnrs-gif.fr PMID:20428338

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

    PubMed Central

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

    2013-01-01

    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. PMID:23482491

  15. Entropy-based measures of in vivo cilia-driven microfluidic mixing derived from quantitative optical imaging

    NASA Astrophysics Data System (ADS)

    Chandrasekera, Kenny; Jonas, Stephan; Bhattacharya, Dipankan; Khokha, Mustafa; Choma, Michael A.

    2012-02-01

    Motile cilia are cellular organelles that project from different epithelial surfaces including respiratory epithelium. They generate directional fluid flow that removes harmful pathogens and particulate matter from the respiratory system. While it has been known that primary ciliary dyskinesia increases the risk of recurrent pulmonary infections, there is now heightened interest in understanding the role that cilia play in a wide-variety of respiratory diseases. Different optical imaging technologies are being investigated to visualize cilia-driven fluid flow, and quantitative image analysis is used to generate measures of ciliary performance. Here, we demonstrate the quantification of in vivo cilia-driven microfluidic mixing using spatial and temporal measures of Shannon information entropy. Using videomicroscopy, we imaged in vivo cilia-driven fluid flow generated by the epidermis of the Xenopus tropicalis embryo. Flow was seeded with either dyes or microparticles. Both spatial and temporal measures of entropy show significant levels of mixing, with maximum entropy measures of ~6.5 (out of a possible range of 0 to 8). Spatial entropy measures showed localization of mixing "hot-spots" and "cold-spots" and temporal measures showed mixing throughout.In sum, entropy-based measures of microfluidic mixing can characterize in vivo cilia-driven fluid flow and hold the potential for better characterization of ciliary dysfunction.

  16. Bio-inspired artificial cilia with magnetic dynamic properties

    NASA Astrophysics Data System (ADS)

    Sun, Leilei; Zheng, Yongmei

    2015-04-01

    Inspired by the structure and properties of natural cilia, we focused on a facile template-free approach to prepare magnetic artificial cilia grown on the substrate (glass, PDMS, or others). In an applied magnetic field, the cilia formed spontaneously and immediately from magnetic nanoparticles and elastomeric polymer in a liquid solvent by bottom-up self-assembly. The length of prepared cilia could be in the scale of millimeter and reach a high aspect ratio of even over 100. We studied the effect of the magnetic strength applied and the size of nanoparticles to get tunable scale of cilia. The cilia show reversibly bending in an external magnetic field and this bending actuation gave some important functions: to transport macroscopic nonmagnetic materials on the cilia and to mix liquids.

  17. Microtubule modifications and stability are altered by cilia perturbation and in cystic kidney disease

    PubMed Central

    Berbari, Nicolas F.; Sharma, Neeraj; Malarkey, Erik B.; Pieczynski, Jay N.; Boddu, Ravindra; Gaertig, Jacek; Guay-Woodford, Lisa; Yoder, Bradley K.

    2013-01-01

    Summary Disruption of the primary cilium is associated with a growing number of human diseases collectively termed ciliopathies. Ciliopathies present with a broad range of clinical features consistent with the near ubiquitous nature of the organelle and its role in diverse signaling pathways throughout development and adult homeostasis. The clinical features associated with cilia dysfunction can include such phenotypes as polycystic kidneys, skeletal abnormalities, blindness, anosmia, and obesity. Although the clinical relevance of the primary cilium is evident, the effects that cilia dysfunction has on the cell and how this contributes to disease remains poorly understood. Here, we show that loss of ciliogenesis genes such as Ift88 and Kif3a lead to increases in post-translational modifications on cytosolic microtubules. This effect was observed in cilia mutant kidney cells grown in vitro and in vivo in cystic kidneys. The hyper-acetylation of microtubules resulting from cilia loss is associated with both altered microtubule stability and increased α-tubulin acetyl-transferase activity. Intriguingly, the effect on microtubules was also evident in renal samples from patients with autosomal recessive polycystic kidneys. These findings indicate that altered microtubule post-translational modifications may influence some of the phenotypes observed in ciliopathies. PMID:23124988

  18. 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

  19. 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. PMID:21331419

  20. Polarised antibranes from Smarr relations

    NASA Astrophysics Data System (ADS)

    Cohen-Maldonado, Diego; Diaz, Juan; Gautason, Fridrik Freyr

    2016-05-01

    We study the backreaction of smeared and localised anti M2-branes placed at the tip of the CGLP background. To this end we derive a Smarr relation for backreacted antibranes at zero and finite temperature. For extremal antibranes we show that if smeared they cannot have regular horizons, whereas localised M2-branes can potentially be regular when polarised into M5-branes, in agreement with the probe result of Klebanov and Pufu. We further discuss antibranes at finite temperature and argue that localised antibrane solutions with regular horizons are not excluded.

  1. Transport of the outer dynein arm complex to cilia requires a cytoplasmic protein Lrrc6.

    PubMed

    Inaba, Yasuko; Shinohara, Kyosuke; Botilde, Yanick; Nabeshima, Ryo; Takaoka, Katsuyoshi; Ajima, Rieko; Lamri, Lynda; Takeda, Hiroyuki; Saga, Yumiko; Nakamura, Tetsuya; Hamada, Hiroshi

    2016-07-01

    Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6(-/-) mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2-IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6(-/-) mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6-dependent mechanism. PMID:27353389

  2. Polarisation of Social Studies Textbooks in Pakistan

    ERIC Educational Resources Information Center

    Zaidi, Syed Manzar Abbas

    2011-01-01

    This article looks at the evolution of the social studies curricula in Pakistan, which are of critical importance in shaping the outlook of many young Pakistanis, who are affected by this polarised discourse. The author argues that this trend of polarisation springing from dynamics of education also effectively contributes to a widening social…

  3. A simple homemade polarised sunglasses test card

    NASA Astrophysics Data System (ADS)

    Bamdad, Farzad

    2016-05-01

    In this article construction of a simple and inexpensive test card which can be used to demonstrate the polarisation ability of sunglasses is described. The card was fabricated simply by using a piece of polariser sheet with one to three layers of cellophane tape fixed on it.

  4. A Simple Homemade Polarised Sunglasses Test Card

    ERIC Educational Resources Information Center

    Bamdad, Farzad

    2016-01-01

    In this article construction of a simple and inexpensive test card which can be used to demonstrate the polarisation ability of sunglasses is described. The card was fabricated simply by using a piece of polariser sheet with one to three layers of cellophane tape fixed on it.

  5. Assembly and dynamics of synthetic cilia

    NASA Astrophysics Data System (ADS)

    Sanchez, Tim

    2012-02-01

    From motility of simple protists to determining the handedness of complex vertebrates, highly conserved eukaryotic cilia and flagella are essential for the reproduction and survival of many biological organisms. Despite extensive studies, the exact mechanism by which individual components coordinate to produce ciliary beating patterns remains unknown. We describe a novel approach towards studying ciliary beating. Instead of deconstructing a fully functional organelle from the top-down, we describe a process by which synthetic cilia-like structures are assembled from the bottom-up. We find that simple mixtures of microtubules, kinesin clusters, and a bundling agent produce spontaneous oscillations in MT bundles, suggesting that self-organized beating may be a generic feature of internally driven bundles. Furthermore, bundles in close proximity spontaneously coordinate their beating to generate metachronal traveling waves, reminiscent of the waves seen in ciliary fields. These findings and future refinements of the system can potentially provide insights into general design principles required for engineering synthetic cilia as well as understanding the biological analogues.

  6. Bardet-Biedl syndrome: Is it only cilia dysfunction?

    PubMed

    Novas, Rossina; Cardenas-Rodriguez, Magdalena; Irigoín, Florencia; Badano, Jose L

    2015-11-14

    Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic disorder, characterized by both congenital and late onset defects. From the analysis of the mutational burden in patients to the functional characterization of the BBS proteins, this syndrome has become a model for both understanding oligogenic patterns of inheritance and the biology of a particular cellular organelle: the primary cilium. Here we briefly review the genetics of BBS to then focus on the function of the BBS proteins, not only in the context of the cilium but also highlighting potential extra-ciliary roles that could be relevant to the etiology of the disorder. Finally, we provide an overview of how the study of this rare syndrome has contributed to the understanding of cilia biology and how this knowledge has informed on the cellular basis of different clinical manifestations that characterize BBS and the ciliopathies. PMID:26231314

  7. 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.

  8. Ion channels and calcium signaling in motile cilia

    PubMed Central

    Doerner, Julia F; Delling, Markus; Clapham, David E

    2015-01-01

    The beating of motile cilia generates fluid flow over epithelia in brain ventricles, airways, and Fallopian tubes. Here, we patch clamp single motile cilia of mammalian ependymal cells and examine their potential function as a calcium signaling compartment. Resting motile cilia calcium concentration ([Ca2+] ~170 nM) is only slightly elevated over cytoplasmic [Ca2+] (~100 nM) at steady state. Ca2+ changes that arise in the cytoplasm rapidly equilibrate in motile cilia. We measured CaV1 voltage-gated calcium channels in ependymal cells, but these channels are not specifically enriched in motile cilia. Membrane depolarization increases ciliary [Ca2+], but only marginally alters cilia beating and cilia-driven fluid velocity within short (~1 min) time frames. We conclude that beating of ependymal motile cilia is not tightly regulated by voltage-gated calcium channels, unlike that of well-studied motile cilia and flagella in protists, such as Paramecia and Chlamydomonas. DOI: http://dx.doi.org/10.7554/eLife.11066.001 PMID:26650848

  9. Sensory functions of motile cilia and implication for bronchiectasis

    PubMed Central

    Jain, Raksha; Javidan-Nejad, Cylen; Alexander-Brett, Jennifer; Horani, Amjad; Cabellon, Michelle C.; Walter, Michael J.; Brody, Steven L.

    2013-01-01

    Cilia are specialized organelles that extend from the surface of cells into the local environment. Airway epithelial cell cilia are motile to provide mucociliary clearance for host defense. On other cells, solitary cilia are specialized to detect chemical or mechanosensory signals. Sensory proteins in motile cilia have recently been identified that detect shear stress, osmotic force, fluid flow, bitter taste and sex hormones. The relationship of sensory function in human motile cilia to disease is now being revealed. One example is polycystin-1 and polycystin-2. As a complex, these proteins function as a flow sensor in cilia and are mutated in autosomal dominant polycystic kidney disease (ADPKD). The polycystins are also expressed in motile cilia of the airways, potentially operating as sensors in the lung. Computed tomography studies from patients with ADPKD revealed radiographic evidence for bronchiectasis, suggesting that polycystin-1 and -2 are important in lung function. The expression of this complex and sensory channel TRPV4, and bitter taste and sex hormones receptors in motile cilia indicate that the cell is wired to interpret environmental cues to regulate cilia beat frequency and other functions. Defective signaling of sensory proteins may result in a ciliopathy that includes lung disease. PMID:22202111

  10. Methods for Studying Movement of Molecules Within Cilia.

    PubMed

    Lechtreck, Karl F

    2016-01-01

    The assembly of cilia and eukaryotic flagella (interchangeable terms) requires the import of numerous proteins from the cell body into the growing organelle. Proteins move into and inside cilia by diffusion and by motor-based intraflagellar transport (IFT). Many aspects of ciliary protein transport such as the distribution of unloading sites and the frequency of transport can be analyzed using direct in vivo imaging of fluorescently tagged proteins. Here, we will describe how to use total internal reflection fluorescence microcopy (TIRFM) to analyze protein transport in the flagella of the unicellular alga Chlamydomonas reinhardtii, a widely used model for cilia and cilia-related disease. PMID:27514917

  11. 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. PMID:25837394

  12. Vector solitons with a uniform polarisation state induced by polarisation filtering in a fibre laser

    SciTech Connect

    Chen Weicheng; Chen Guojie; Han Dingan; Xie Jianing

    2013-06-30

    A fibre laser with a SESAM as a passive mode-locker is constructed for obtaining a vector soliton with the Kelly sidebands. The analysis of the peculiarities of the sidebands shows that the polarisation states are nonuniform across the entire pulse spectral profile from the leading edge to the trailing edge. Polarisation filtering effect is proposed to obtain a vector soliton with a uniform polarisation state. It is shown that during the polarisation filtering by a polariser incorporated into the laser cavity, the spectral width of the vector solitons gradually broadens and the pulse power decreases. It is found that at a maximum spectral width and a minimum pulse power, vector solitons with a uniform polarisation state are generated. (nonlinear optical phenomena)

  13. ATR promotes cilia signalling: links to developmental impacts.

    PubMed

    Stiff, Tom; Casar Tena, Teresa; O'Driscoll, Mark; Jeggo, Penny A; Philipp, Melanie

    2016-04-15

    Mutations in ATR(ataxia telangiectasia and RAD3-related) cause Seckel syndrome (ATR-SS), a microcephalic primordial dwarfism disorder. Hitherto, the clinical manifestation of ATR deficiency has been attributed to its canonical role in DNA damage response signalling following replication fork stalling/collapse. Here, we show that ATR regulates cilia-dependent signalling in a manner that can be uncoupled from its function during replication. ATR-depleted or patient-derived ATR-SS cells form cilia of slightly reduced length but are dramatically impaired in cilia-dependent signalling functions, including growth factor and Sonic hedgehog signalling. To better understand the developmental impact of ATR loss of function, we also used zebrafish as a model. Zebrafish embryos depleted of Atr resembled ATR-SS morphology, showed a modest but statistically significant reduction in cilia length and other morphological features indicative of cilia dysfunction. Additionally, they displayed defects in left-right asymmetry including ambiguous expression of southpaw, incorrectly looped hearts and randomized localization of internal organs including the pancreas, features typically conferred by cilia dysfunction. Our findings reveal a novel role for ATR in cilia signalling distinct from its canonical function during replication and strengthen emerging links between cilia function and development. PMID:26908596

  14. Left-right asymmetry: cilia and calcium revisited.

    PubMed

    Blum, Martin; Vick, Philipp

    2015-03-01

    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. PMID:25734272

  15. Cilia and Nuclear Pore Proteins: Pore No More?

    PubMed

    Obado, Samson O; Rout, Michael P

    2016-09-12

    Nuclear pore proteins at the base of cilia were thought to regulate transport into cilia. In this issue of Developmental Cell, Del Viso et al. (2016) challenge this view, showing instead that pore proteins localize to ciliary basal bodies and that their perturbation leads to congenital heart disease. PMID:27623377

  16. ATR promotes cilia signalling: links to developmental impacts

    PubMed Central

    Stiff, Tom; Casar Tena, Teresa; O'Driscoll, Mark; Jeggo, Penny A.; Philipp, Melanie

    2016-01-01

    Mutations in ATR (ataxia telangiectasia and RAD3-related) cause Seckel syndrome (ATR-SS), a microcephalic primordial dwarfism disorder. Hitherto, the clinical manifestation of ATR deficiency has been attributed to its canonical role in DNA damage response signalling following replication fork stalling/collapse. Here, we show that ATR regulates cilia-dependent signalling in a manner that can be uncoupled from its function during replication. ATR-depleted or patient-derived ATR-SS cells form cilia of slightly reduced length but are dramatically impaired in cilia-dependent signalling functions, including growth factor and Sonic hedgehog signalling. To better understand the developmental impact of ATR loss of function, we also used zebrafish as a model. Zebrafish embryos depleted of Atr resembled ATR-SS morphology, showed a modest but statistically significant reduction in cilia length and other morphological features indicative of cilia dysfunction. Additionally, they displayed defects in left-right asymmetry including ambiguous expression of southpaw, incorrectly looped hearts and randomized localization of internal organs including the pancreas, features typically conferred by cilia dysfunction. Our findings reveal a novel role for ATR in cilia signalling distinct from its canonical function during replication and strengthen emerging links between cilia function and development. PMID:26908596

  17. First Polarised Light with the NIKA Camera

    NASA Astrophysics Data System (ADS)

    Ritacco, A.; Adam, R.; Adane, A.; Ade, P.; André, P.; Beelen, A.; Belier, B.; Benoît, A.; Bideaud, A.; Billot, N.; Bourrion, O.; Calvo, M.; Catalano, A.; Coiffard, G.; Comis, B.; D'Addabbo, A.; Désert, F.-X.; Doyle, S.; Goupy, J.; Kramer, C.; Leclercq, S.; Macías-Pérez, J. F.; Martino, J.; Mauskopf, P.; Maury, A.; Mayet, F.; Monfardini, A.; Pajot, F.; Pascale, E.; Perotto, L.; Pisano, G.; Ponthieu, N.; Rebolo-Iglesias, M.; Revéret, V.; Rodriguez, L.; Savini, G.; Schuster, K.; Sievers, A.; Thum, C.; Triqueneaux, S.; Tucker, C.; Zylka, R.

    2015-10-01

    NIKA is a dual-band camera operating with 315 frequency multiplexed LEKIDs cooled at 100 mK. NIKA is designed to observe the sky in intensity and polarisation at 150 and 260 GHz from the IRAM 30-m telescope. It is a test-bench for the final NIKA2 camera. The incoming linear polarisation is modulated at four times the mechanical rotation frequency by a warm rotating multi-layer half- wave plate. Then, the signal is analyzed by a wire grid and finally absorbed by the lumped element kinetic inductance detectors (LEKIDs). The small time constant (< 1 ms ) of the LEKIDs combined with the modulation of the HWP enables the quasi-simultaneous measurement of the three Stokes parameters I, Q, U, representing linear polarisation. In this paper, we present the results of recent observational campaigns demonstrating the good performance of NIKA in detecting polarisation at millimeter wavelength.

  18. First Polarised Light with the NIKA Camera

    NASA Astrophysics Data System (ADS)

    Ritacco, A.; Adam, R.; Adane, A.; Ade, P.; André, P.; Beelen, A.; Belier, B.; Benoît, A.; Bideaud, A.; Billot, N.; Bourrion, O.; Calvo, M.; Catalano, A.; Coiffard, G.; Comis, B.; D'Addabbo, A.; Désert, F.-X.; Doyle, S.; Goupy, J.; Kramer, C.; Leclercq, S.; Macías-Pérez, J. F.; Martino, J.; Mauskopf, P.; Maury, A.; Mayet, F.; Monfardini, A.; Pajot, F.; Pascale, E.; Perotto, L.; Pisano, G.; Ponthieu, N.; Rebolo-Iglesias, M.; Revéret, V.; Rodriguez, L.; Savini, G.; Schuster, K.; Sievers, A.; Thum, C.; Triqueneaux, S.; Tucker, C.; Zylka, R.

    2016-08-01

    NIKA is a dual-band camera operating with 315 frequency multiplexed LEKIDs cooled at 100 mK. NIKA is designed to observe the sky in intensity and polarisation at 150 and 260 GHz from the IRAM 30-m telescope. It is a test-bench for the final NIKA2 camera. The incoming linear polarisation is modulated at four times the mechanical rotation frequency by a warm rotating multi-layer half- wave plate. Then, the signal is analyzed by a wire grid and finally absorbed by the lumped element kinetic inductance detectors (LEKIDs). The small time constant (<1 ms ) of the LEKIDs combined with the modulation of the HWP enables the quasi-simultaneous measurement of the three Stokes parameters I, Q, U, representing linear polarisation. In this paper, we present the results of recent observational campaigns demonstrating the good performance of NIKA in detecting polarisation at millimeter wavelength.

  19. An ultrafast carbon nanotube terahertz polarisation modulator

    NASA Astrophysics Data System (ADS)

    Docherty, Callum J.; Stranks, Samuel D.; Habisreutinger, Severin N.; Joyce, Hannah J.; Herz, Laura M.; Nicholas, Robin J.; Johnston, Michael B.

    2014-05-01

    We demonstrate ultrafast modulation of terahertz radiation by unaligned optically pumped single-walled carbon nanotubes. Photoexcitation by an ultrafast optical pump pulse induces transient terahertz absorption in nanowires aligned parallel to the optical pump. By controlling the polarisation of the optical pump, we show that terahertz polarisation and modulation can be tuned, allowing sub-picosecond modulation of terahertz radiation. Such speeds suggest potential for semiconductor nanowire devices in terahertz communication technologies.

  20. Polarisation losses in a ring prism cavity

    SciTech Connect

    Kuryatov, V N; Sokolov, A L

    2000-02-28

    The polarisation losses in a ring cavity, formed by total-internal-reflection prisms, were analysed. All the sources of the polarisation losses are indicated and expressions for their calculation are presented. The limit to loss reduction in cavities of this kind, set by the difference between the radii of curvature of the radiation wavefront and of the refracting prism faces, was determined. (laser gyroscopes)

  1. An ultrafast carbon nanotube terahertz polarisation modulator

    SciTech Connect

    Docherty, Callum J.; Stranks, Samuel D.; Habisreutinger, Severin N.; Joyce, Hannah J.; Herz, Laura M.; Nicholas, Robin J.; Johnston, Michael B.

    2014-05-28

    We demonstrate ultrafast modulation of terahertz radiation by unaligned optically pumped single-walled carbon nanotubes. Photoexcitation by an ultrafast optical pump pulse induces transient terahertz absorption in nanowires aligned parallel to the optical pump. By controlling the polarisation of the optical pump, we show that terahertz polarisation and modulation can be tuned, allowing sub-picosecond modulation of terahertz radiation. Such speeds suggest potential for semiconductor nanowire devices in terahertz communication technologies.

  2. Dynamic nuclear polarisation of diamond

    NASA Astrophysics Data System (ADS)

    High, Grant Lysle

    Chapter one reviews the reported literature on the NMR of diamond. This signal consists of a single line at 39 ppm from TMS and two hyperfine lines due to 13C interactions. The second chapter introduces the apparatus used in this study. The availability of this excellently equipped laboratory presented a unique opportunity to perform this investigation. Chapter three outlines the experimental techniques used as well as the manner in which the acquired data was processed. The fourth chapter presents an overview of the most common defects found in diamond. Proposed models of these defects are presented and the EPR spectra displayed. The methods developed to determine the paramagnetic impurity concentration, from the EPR line width and the spin-spin relaxation times are presented in the fifth chapter. The line width gives the total paramagnetic impurity concentration to about 10 ppm. The spin-spin relaxation time allows the determination of paramagnetic impurity concentrations, to much lower levels. This information was used in the explanation of the relaxation behaviour for the diamonds investigated. The temperature dependence of the paramagnetic electron relaxation times is reported in the sixth chapter. The results obtained are consistent with the findings in prior work that P1 impurities are typical, Jahn Teller centres. Two diamonds, however, display trends that depart from this theory. It was found in these experiments that, bar thermal expansion effects, the spin-spin relaxation time is independent of temperature. The seventh chapter deals with the solid state and thermal mixing effects. The relevant theory, results obtained and a discussion of these results, are presented. The effect of various parameters on the polarisation rates and the 13C signal enhancement are investigated. Finally the effect of applying the DNP treatment on the central and hyperfine lines is discussed. The pulsed DNP process is presented in the eighth chapter. The relevant theory, the

  3. A new polarisation amplitude bias reduction method.

    NASA Astrophysics Data System (ADS)

    Vidal, M.; Leahy, J. P.; Dickinson, C.

    2016-06-01

    Polarisation amplitude estimation is affected by a positive noise bias, particularly important in regions with low signal-to-noise ratio (SNR). We present a new approach to correct for this bias in the case there is additional information about the polarisation angle. We develop the `known-angle estimator' that works in the special case when there is an independent and high signal-to-noise ratio (≳ 2σ) measurement of the polarisation angle. It is derived for the general case where the uncertainties in the Q, U Stokes parameters are not symmetric. This estimator completely corrects for the polarisation bias if the polarisation angle is perfectly known. In the realistic case, where the angle template has uncertainties, a small residual bias remains, but that is shown to be much smaller that the one left by other classical estimators. We also test our method with more realistic data, using the noise properties of the three lower frequency maps of WMAP. In this case, the known-angle estimator also produces better results than methods that do not include the angle information. This estimator is therefore useful in the case where the polarisation angle is expected to be constant over different data sets with different SNR.

  4. Computation of the internal forces in cilia: application to ciliary motion, the effects of viscosity, and cilia interactions.

    PubMed

    Gueron, S; Levit-Gurevich, K

    1998-04-01

    This paper presents a simple and reasonable method for generating a phenomenological model of the internal mechanism of cilia. The model uses a relatively small number of parameters whose values can be obtained by fitting to ciliary beat shapes. Here, we use beat patterns observed in Paramecium. The forces that generate these beats are computed and fit to a simple functional form called the "engine." This engine is incorporated into a recently developed hydrodynamic model that accounts for interactions between neighboring cilia and between the cilia and the surface from which they emerge. The model results are compared to data on ciliary beat patterns of Paramecium obtained under conditions where the beats are two-dimensional. Many essential features of the motion, including several properties that are not built in explicitly, are shown to be captured. In particular, the model displays a realistic change in beat pattern and frequency in response to increased viscosity and to the presence of neighboring cilia in configurations such as rows of cilia and two-dimensional arrays of cilia. We found that when two adjacent model cilia start beating at different phases they become synchronized within several beat periods, as observed in experiments where two flagella are brought into close proximity. Furthermore, examination of various multiciliary configurations shows that an approximately antiplectic wave pattern evolves autonomously. This modeling evidence supports earlier conjectures that metachronism may occur, at least partially, as a self-organized phenomenon due to hydrodynamic interactions between neighboring cilia. PMID:9545031

  5. Cilia-Associated Genes Play Differing Roles in Aminoglycoside-Induced Hair Cell Death in Zebrafish

    PubMed Central

    Stawicki, Tamara M.; Hernandez, Liana; Esterberg, Robert; Linbo, Tor; Owens, Kelly N.; Shah, Arish N.; Thapa, Nihal; Roberts, Brock; Moens, Cecilia B.; Rubel, Edwin W.; Raible, David W.

    2016-01-01

    Hair cells possess a single primary cilium, called the kinocilium, early in development. While the kinocilium is lost in auditory hair cells of most species it is maintained in vestibular hair cells. It has generally been believed that the primary role of the kinocilium and cilia-associated genes in hair cells is in the establishment of the polarity of actin-based stereocilia, the hair cell mechanotransduction apparatus. Through genetic screening and testing of candidate genes in zebrafish (Danio rerio) we have found that mutations in multiple cilia genes implicated in intraflagellar transport (dync2h1, wdr35, ift88, and traf3ip), and the ciliary transition zone (cc2d2a, mks1, and cep290) lead to resistance to aminoglycoside-induced hair cell death. These genes appear to have differing roles in hair cells, as mutations in intraflagellar transport genes, but not transition zone genes, lead to defects in kinocilia formation and processes dependent upon hair cell mechanotransduction activity. These mutants highlight a novel role of cilia-associated genes in hair cells, and provide powerful tools for further study. PMID:27207957

  6. Cilia-Associated Genes Play Differing Roles in Aminoglycoside-Induced Hair Cell Death in Zebrafish.

    PubMed

    Stawicki, Tamara M; Hernandez, Liana; Esterberg, Robert; Linbo, Tor; Owens, Kelly N; Shah, Arish N; Thapa, Nihal; Roberts, Brock; Moens, Cecilia B; Rubel, Edwin W; Raible, David W

    2016-01-01

    Hair cells possess a single primary cilium, called the kinocilium, early in development. While the kinocilium is lost in auditory hair cells of most species it is maintained in vestibular hair cells. It has generally been believed that the primary role of the kinocilium and cilia-associated genes in hair cells is in the establishment of the polarity of actin-based stereocilia, the hair cell mechanotransduction apparatus. Through genetic screening and testing of candidate genes in zebrafish (Danio rerio) we have found that mutations in multiple cilia genes implicated in intraflagellar transport (dync2h1, wdr35, ift88, and traf3ip), and the ciliary transition zone (cc2d2a, mks1, and cep290) lead to resistance to aminoglycoside-induced hair cell death. These genes appear to have differing roles in hair cells, as mutations in intraflagellar transport genes, but not transition zone genes, lead to defects in kinocilia formation and processes dependent upon hair cell mechanotransduction activity. These mutants highlight a novel role of cilia-associated genes in hair cells, and provide powerful tools for further study. PMID:27207957

  7. An age of enlightenment for cilia: The FASEB summer research conference on the "Biology of Cilia and Flagella".

    PubMed

    Tran, Pamela V; Lechtreck, Karl F

    2016-01-15

    From July 19-24, 2015, 169 clinicians and basic scientists gathered in the vertiginous heights of Snowmass, Colorado (2502 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

  8. Structural defects in cilia of the choroid plexus, subfornical organ and ventricular ependyma are associated with ventriculomegaly

    PubMed Central

    2012-01-01

    Background Hydrocephalus is a heterogeneous disorder with multiple etiologies that are not yet fully understood. Animal models have implicated dysfunctional cilia of the ependyma and choroid plexus in the development of the disorder. In this report, we sought to determine the origin of the ventriculomegaly in four Bardet Biedl syndrome (BBS) mutant mouse strains as models of a ciliopathy. Methods Evans Blue dye was injected into the lateral ventricle of wild- type and BBS mutant mice to determine whether obstruction of intra- or extra-ventricular CSF flow contributed to ventriculomegaly. Transmission electron microscopy (TEM) was used to examine the ultrastructure of the choroid plexus, subfornical organ (SFO), subcommisural organ (SCO), and ventricular ependyma to evaluate their ultrastructure and the morphology of their primary and motile cilia. Results and discussion No obstruction of intra- or extra-ventricular CSF flow was observed, implying a communicating form of hydrocephalus in BBS mutant mice. TEM analyses of the mutants showed no evidence of choroidal papillomas or breakdown of the blood:CSF barrier. In contrast, structural defects were observed in a subpopulation of cilia lining the choroid plexus, SFO, and ventricular ependyma. These included disruptions of the microtubular structure of the axoneme and the presence of electron-dense vesicular-like material along the ciliary shaft and at the tips of cilia. Conclusions Abnormalities in cilia structure and function have the potential to influence ciliary intraflagellar transport (IFT), cilia maintenance, protein trafficking, and regulation of CSF production. Ciliary structural defects are the only consistent pathological features associated with CSF-related structures in BBS mutant mice. These defects are observed from an early age, and may contribute to the underlying pathophysiology of ventriculomegaly. PMID:23046663

  9. Biomimetic cilia arrays generate simultaneous pumping and mixing regimes

    PubMed Central

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

    2010-01-01

    Living systems employ cilia to control and to sense the flow of fluids for many purposes, such as pumping, locomotion, feeding, and tissue morphogenesis. Beyond their use in biology, functional arrays of artificial cilia have been envisaged as a potential biomimetic strategy for inducing fluid flow and mixing in lab-on-a-chip devices. Here we report on fluid transport produced by magnetically actuated arrays of biomimetic cilia whose size approaches that of their biological counterparts, a scale at which advection and diffusion compete to determine mass transport. Our biomimetic cilia recreate the beat shape of embryonic nodal cilia, simultaneously generating two sharply segregated regimes of fluid flow: Above the cilia tips their motion causes directed, long-range fluid transport, whereas below the tips we show that the cilia beat generates an enhanced diffusivity capable of producing increased mixing rates. These two distinct types of flow occur simultaneously and are separated in space by less than 5 μm, approximately 20% of the biomimetic cilium length. While this suggests that our system may have applications as a versatile microfluidics device, we also focus on the biological implications of our findings. Our statistical analysis of particle transport identifying an enhanced diffusion regime provides novel evidence for the existence of mixing in ciliated systems, and we demonstrate that the directed transport regime is Poiseuille–Couette flow, the first analytical model consistent with biological measurements of fluid flow in the embryonic node. PMID:20798342

  10. Fetus Sound Stimulation: Cilia Memristor Effect of Signal Transduction

    PubMed Central

    Jankovic-Raznatovic, Svetlana; Dragojevic-Dikic, Svetlana; Rakic, Snezana; Nikolic, Branka; Plesinac, Snezana; Tasic, Lidija; Perisic, Zivko; Sovilj, Mirjana; Adamovic, Tatjana; Koruga, Djuro

    2014-01-01

    Background. This experimental study evaluates fetal middle cerebral artery (MCA) circulation after the defined prenatal acoustical stimulation (PAS) and the role of cilia in hearing and memory and could explain signal transduction and memory according to cilia optical-acoustical properties. Methods. PAS was performed twice on 119 no-risk term pregnancies. We analyzed fetal MCA circulation before, after first and second PAS. Results. Analysis of the Pulsatility index basic (PIB) and before PAS and Pulsatility index reactive after the first PAS (PIR 1) shows high statistical difference, representing high influence on the brain circulation. Analysis of PIB and Pulsatility index reactive after the second PAS (PIR 2) shows no statistical difference. Cilia as nanoscale structure possess magnetic flux linkage that depends on the amount of charge that has passed between two-terminal variable resistors of cilia. Microtubule resistance, as a function of the current through and voltage across the structure, leads to appearance of cilia memory with the “memristor” property. Conclusion. Acoustical and optical cilia properties play crucial role in hearing and memory processes. We suggest that fetuses are getting used to sound, developing a kind of memory patterns, considering acoustical and electromagnetically waves and involving cilia and microtubules and try to explain signal transduction. PMID:24719851

  11. New Measurements of Polarised Light from Exoplanets

    NASA Astrophysics Data System (ADS)

    Bott, Kimberly Marie; Cotton, Daniel; Kedziora-Chudczer, Lucyna; Bailey, Jeremy

    2015-08-01

    Detections of polarised light from exoplanets are an important expansion of exoplanet studies, as they provide a complimentary and advantageous diagnostic to the other characterisation methods. However some of the earliest claimed detections of polarised light from exoplanets are disputed.The High Precision Polarimetric Instrument (HIPPI) is currently the highest sensitivity astronomical polarimeter (Bailey 2015) in the world at 4.3 ppm or better precision, and has been used to observe exoplanet systems including the disputed first detection source (Berdyugina 2008, Wiktorowicz 2009, Berdyugina 2011), HD189733b. HIPPI is designed for best sensitivity in blue light where Rayleigh scattering would produce a strong signal for hot Jupiter planets (Burrows 2008). These observations, taken at the Anglo-Australian Telescope in 2014 and 2015, are compared to models from a polarised light adaptation of the Versatile Software for the Transfer of Atmospheric Radiation (VSTAR).Our new, independent Hot Jupiter polarised light detections are discussed, including the physical sources of the detected polarised light, as well as additional contributing factors (e.g. debris, ISM anomalies). For HD 189733b we use available visible and near infrared transit and polarimetric data to create and interpret a planetary portrait using VSTAR.

  12. Cilia-associated bacteria in fatal Bordetella bronchiseptica pneumonia of dogs and cats.

    PubMed

    Taha-Abdelaziz, Khaled; Bassel, Laura L; Harness, Melanie L; Clark, Mary Ellen; Register, Karen B; Caswell, Jeff L

    2016-07-01

    Bordetella bronchiseptica frequently causes nonfatal tracheobronchitis, but its role in fatal pneumonia is less recognized. Our study evaluated histologic identification of cilia-associated bacteria as a method for diagnosis of B. bronchiseptica pneumonia. Cases of fatal bronchopneumonia were studied retrospectively, excluding neonates and cases of aspiration pneumonia, minor lung lesions, or autolysis. The study population comprised 36 canine and 31 feline cases of bronchopneumonia. B. bronchiseptica was identified in 8 of 36 canine and 14 of 31 feline cases based on immunohistochemistry (IHC) using serum from a rabbit hyperimmunized with pertactin, PCR testing (Fla2/Fla12), and/or bacterial culture data when available. Of these, IHC was positive in 4 canine and 7 feline cases, PCR was positive in 8 canine and 14 feline cases, and B. bronchiseptica was isolated in 2 of 5 canine and 3 of 9 feline cases tested. Examination of histologic sections stained with hematoxylin and eosin revealed bronchial cilia-associated bacteria in 4 of 36 canine and 5 of 31 feline cases; these were all positive by IHC and PCR. The presence of cilia-associated bacteria had been noted in the pathology report for only 2 of these 9 cases. Thus, the presence of cilia-associated bacteria seems frequently overlooked by pathologists, but is a diagnostically significant feature of B. bronchiseptica pneumonia. A specific diagnosis of B. bronchiseptica pneumonia is important because it suggests primary or opportunistic bacterial pneumonia rather than aspiration pneumonia, and because of the risk of animal-to-animal transmission of B. bronchiseptica, the availability of vaccines for disease prevention, and the potential zoonotic risk to immunocompromised pet owners. PMID:27178716

  13. Linking the Primary Cilium to Cell Migration in Tissue Repair and Brain Development

    PubMed Central

    Veland, Iben Rønn; Lindbæk, Louise; Christensen, Søren Tvorup

    2014-01-01

    Primary cilia are unique sensory organelles that coordinate cellular signaling networks in vertebrates. Inevitably, defects in the formation or function of primary cilia lead to imbalanced regulation of cellular processes that causes multisystemic disorders and diseases, commonly known as ciliopathies. Mounting evidence has demonstrated that primary cilia coordinate multiple activities that are required for cell migration, which, when they are aberrantly regulated, lead to defects in organogenesis and tissue repair, as well as metastasis of tumors. Here, we present an overview on how primary cilia may contribute to the regulation of the cellular signaling pathways that control cyclic processes in directional cell migration. PMID:26955067

  14. The significance of ultrastructural abnormalities of human cilia.

    PubMed

    Fox, B; Bull, T B; Makey, A R; Rawbone, R

    1981-12-01

    The electronmicroscopic structure of cilia was studied from the inferior turbinate of the nose in 22 adults, and in 84 biopsies from the bronchial tree of 40 adults. The incidence of compound cilia and abnormal microtubular structures was assessed. There were significant variations in the incidence of abnormalities in different parts of the airways and even within different areas of the same electronmicroscopic section. The focal nature of differences in structure of cilia indicate that abnormalities found in a single biopsy do not necessarily reflect a generalized change in the bronchial tree. Thus, such a finding should not be used as evidence that the abnormalities of cilia are the cause of decrease in mucociliary clearance or that they play a role in the pathogenesis of bronchiectasis and sinusitis. PMID:7307613

  15. 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.

  16. Using Xenopus Skin to Study Cilia Development and Function

    PubMed Central

    Werner, Michael E.; Mitchell, Brian J.

    2015-01-01

    Cilia are prevalent biological structures that are important for cell signaling and for generating fluid flow (or motility). Cilia are found throughout biology from single-celled organisms to vertebrates, and many model systems have been employed for their analysis. Here, we describe the use of Xenopus larval skin as a system for the study of ciliogenesis and ciliary function. In particular, we describe basic molecular and embryological manipulations and imaging techniques that have proven particularly useful for understanding the polarized beating of cilia and the generation of directed fluid flow (Werner & Mitchell, 2012). However, these same tools have the potential to benefit a large number of cilia-related biological questions. PMID:23522471

  17. Microscale imaging of cilia-driven fluid flow

    PubMed Central

    Huang, Brendan K.; Choma, Michael A.

    2015-01-01

    Cilia-driven fluid flow is important for multiple processes in the body, including respiratory mucus clearance, gamete transport in the oviduct, right-left patterning in the embryonic node, and cerebrospinal fluid circulation. Multiple imaging techniques have been applied towards quantifying ciliary flow. Here we review common velocimetry methods of quantifying fluid flow. We then discuss four important optical modalities, including light microscopy, epifluorescence, confocal microscopy, and optical coherence tomography, that have been used to investigate cilia-driven flow. PMID:25417211

  18. 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.

  19. Endothelial Cilia Are Essential for Developmental Vascular Integrity in Zebrafish

    PubMed Central

    Kallakuri, Sowjanya; Yu, Jianxin A.; Li, Jade; Li, Yuanyuan; Weinstein, Brant M.; Nicoli, Stefania

    2015-01-01

    The cilium is a signaling platform of the vertebrate cell. It has a critical role in polycystic kidney disease and nephronophthisis. Cilia have been detected on endothelial cells, but the function of these organelles in the vasculature remains incompletely defined. In this study, using genetic and chemical genetic tools in the model organism zebrafish, we reveal an essential role of cilia in developmental vascular integrity. Embryos expressing mutant intraflagellar transport genes, which are essential and specific for cilia biogenesis, displayed increased risk of developmental intracranial hemorrhage, whereas the morphology of the vasculature remained normal. Moreover, cilia were present on endothelial cells in the developing zebrafish vasculature. We further show that the involvement of cilia in vascular integrity is endothelial autonomous, because endothelial-specific re-expression of intraflagellar transport genes in respective mutants rescued the intracranial hemorrhage phenotype. Finally, whereas inhibition of Hedgehog signaling increased the risk of intracranial hemorrhage in ciliary mutants, activation of the pathway rescued this phenotype. In contrast, embryos expressing an inactivating mutation in pkd2, one of two autosomal dominant cystic kidney disease genes, did not show increased risk of developmental intracranial hemorrhage. These results suggest that Hedgehog signaling is a major mechanism for this novel role of endothelial cilia in establishing vascular integrity. PMID:25214579

  20. 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. PMID:26439855

  1. Generating conditional mutants to analyze ciliary functions: the use of Cre-lox technology to disrupt cilia in specific organs.

    PubMed

    O'Connor, Amber K; Kesterson, Robert A; Yoder, Bradley K

    2009-01-01

    The list of human disordered associated with cilia dysfunction, the ciliopathies, continues to highlight the importance of understanding the many roles of the long overlooked primary cilium. Much of the insights into the clinical importance of the cilium have come from analyses in model organisms, especially the mouse. However, the early embryonic lethality and severe developmental defects associated with cilia disruption has hindered progress in exploring cilia functions in late development or in adult tissues. This hurdle is being surmounted through the use of conditional alleles of genes encoding ciliary proteins and Cre deletor lines with inducible Cre activity or with lines expressing Cre in a cell-type-specific manner. Results from these approaches are providing important insights into the diverse array of cellular and tissue activities regulated by the cilium. Here we provide a recent account of the Cre/lox strategy. The generation and use of well-designed conditional alleles, as well as careful manipulation of embryonic stem cells are discussed. We also provide specific examples to illustrate the use of Cre/lox approaches to evaluate ciliary function in several tissues. With the recent characterization of multiple cilia proteomes along with efforts of several consortia to generate conditional alleles of all genes in the mouse, further use of conditional mutation approaches promise to yield many advances and surprises as we explore the functions of this increasingly complex organelle. PMID:20409823

  2. A high-resolution morphological and ultrastructural map of anterior sensory cilia and glia in Caenorhabditis elegans

    PubMed Central

    Doroquez, David B; Berciu, Cristina; Anderson, James R; Sengupta, Piali; Nicastro, Daniela

    2014-01-01

    Many primary sensory cilia exhibit unique architectures that are critical for transduction of specific sensory stimuli. Although basic ciliogenic mechanisms are well described, how complex ciliary structures are generated remains unclear. Seminal work performed several decades ago provided an initial but incomplete description of diverse sensory cilia morphologies in C. elegans. To begin to explore the mechanisms that generate these remarkably complex structures, we have taken advantage of advances in electron microscopy and tomography, and reconstructed three-dimensional structures of fifty of sixty sensory cilia in the C. elegans adult hermaphrodite at high resolution. We characterize novel axonemal microtubule organization patterns, clarify structural features at the ciliary base, describe new aspects of cilia–glia interactions, and identify structures suggesting novel mechanisms of ciliary protein trafficking. This complete ultrastructural description of diverse cilia in C. elegans provides the foundation for investigations into underlying ciliogenic pathways, as well as contributions of defined ciliary structures to specific neuronal functions. DOI: http://dx.doi.org/10.7554/eLife.01948.001 PMID:24668170

  3. 9 + 0 and 9 + 2 cilia are randomly dispersed in the mouse node.

    PubMed

    Odate, Toru; Takeda, Sen; Narita, Keishi; Kawahara, Toru

    2016-04-01

    The initial determination of left-right asymmetry is an essential process in embryonic development. In mouse embryo, cilia in the node play an important role generating the nodal flow that subsequently triggers left-right determination in the embryo. Although nodal cilia have historically been thought to have a 9 + 0 axonemal configuration, the existence of 9 + 2 cilia has been reported so far. Because the distribution of those two types of cilia within the node has not yet been reported, we assessed the arrangement of 9 + 0 and 9 + 2 cilia in the node. In this study, we concluded that most of the nodal cilia were 9 + 0 in structure and there were much fewer 9 + 2 cilia than 9 + 0 cilia. Furthermore, the two types of cilia were randomly distributed in the node with no regularity. In addition, we studied the embryonic origin of the crown cells surrounding the node to better understand their identity. PMID:26520785

  4. Polarisation modulation in X-ray binaries

    NASA Astrophysics Data System (ADS)

    Ingram, Adam; Maccarone, Thomas

    2016-07-01

    X-ray polarimetry promises to provide a powerful new lever arm for studying accretion onto black holes with the next generation of X-ray telescopes. I will discuss how polarisation can be used to help constrain the physical origin of quasi-periodic oscillations (QPOs) observed in the X-ray light curves of accreting black holes. QPOs may be signatures of the frame dragging effect: in General Relativity, a spinning black hole twists up the surrounding space-time, causing vertical precession of nearby orbits. In the truncated disc / precessing inner flow model, the entire inner accretion flow precesses as a solid body causing a modulation in the X-ray flux through solid angle and Doppler effects. This model also predicts the observed polarisation of the X-ray signal to vary quasi-periodically. I will summarise our work to model the polarisation signal from a precessing accretion flow, starting with simple assumptions about the emission mechanism but taking General Relativity fully into account. We find that it should be possible to measure the predicted modulation in polarisation degree for a reasonable region of parameter space with a polarimeter capable of detecting ~60 counts per second from a bright black hole binary. I will also show that sensitivity can be greatly improved by correlating the signal with a high count rate reference band signal.

  5. Optimal polarisation equations in FLRW universes

    SciTech Connect

    Tram, Thomas; Lesgourgues, Julien E-mail: Julien.Lesgourgues@cern.ch

    2013-10-01

    This paper presents the linearised Boltzmann equation for photons for scalar, vector and tensor perturbations in flat, open and closed FLRW cosmologies. We show that E- and B-mode polarisation for all types can be computed using only a single hierarchy. This was previously shown explicitly for tensor modes in flat cosmologies but not for vectors, and not for non-flat cosmologies.

  6. Midbody remnant licenses primary cilia formation in epithelial cells.

    PubMed

    Ott, Carolyn M

    2016-08-01

    Tethered midbody remnants dancing across apical microvilli, encountering the centrosome, and beckoning forth a cilium-who would have guessed this is how polarized epithelial cells coordinate the end of mitosis and the beginning of ciliogenesis? New evidence from Bernabé-Rubio et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201601020) supports this emerging model. PMID:27482049

  7. The coiled-coil domain containing protein CCDC151 is required for the function of IFT-dependent motile cilia in animals.

    PubMed

    Jerber, Julie; Baas, Dominique; Soulavie, Fabien; Chhin, Brigitte; Cortier, Elisabeth; Vesque, Christine; Thomas, Joëlle; Durand, Bénédicte

    2014-02-01

    Cilia are evolutionarily conserved organelles endowed with essential physiological and developmental functions. In humans, disruption of cilia motility or signaling leads to complex pleiotropic genetic disorders called ciliopathies. Cilia motility requires the assembly of multi-subunit motile components such as dynein arms, but mechanisms underlying their assembly pathway and transport into the axoneme are still largely unknown. We identified a previously uncharacterized coiled-coil domain containing protein CCDC151, which is evolutionarily conserved in motile ciliated species and shares ancient features with the outer dynein arm-docking complex 2 of Chlamydomonas. In Drosophila, we show that CG14127/CCDC151 is associated with motile intraflagellar transport (IFT)-dependent cilia and required for geotaxis behavior of adult flies. In zebrafish, Ccdc151 is expressed in tissues with motile cilia, and morpholino-induced depletion of Ccdc151 leads to left-right asymmetry defects and kidney cysts. We demonstrate that Ccdc151 is required for proper motile function of cilia in the Kupffer's vesicle and in the pronephros by controlling dynein arm assembly, showing that Ccdc151 is a novel player in the control of IFT-dependent dynein arm assembly in animals. However, we observed that CCDC151 is also implicated in other cellular functions in vertebrates. In zebrafish, ccdc151 is involved in proper orientation of cell divisions in the pronephros and genetically interacts with prickle1 in this process. Furthermore, knockdown experiments in mammalian cells demonstrate that CCDC151 is implicated in the regulation of primary cilium length. Hence, CCDC151 is required for motile cilia function in animals but has acquired additional non-motile functions in vertebrates. PMID:24067530

  8. Oscillations of Eukaryotic Cilia and Flagella

    NASA Astrophysics Data System (ADS)

    Gopinath, Arvind; Mahadevan, Lakshminarayanan

    2006-11-01

    The undulating beat of eukaryotic flagella and cilia produces forces that move cells and cause locomotion. The timing mechanisms that generate these periodic undulations are still mysterious and the question of how these oscillations arise is still a subject of much research - both experimental and theoretical. Recent experimental results on paralyzed and reconstituted flagella offer new insight into the dynamical mechanisms that could result in sustained waveform generation. Motivated by these recent experimental results we propose a model that mimics the flagellar structure as motor driven elastic, inextensible filaments. We hypothesize that the oscillations arise due to motor (dynein) driven, constrained, relative sliding of parts of the flagella. The dynamical equations describing the evolution of the populations of attached and detached motors is actively coupled to the local configuration as well as local sliding velocities via strain and configuration dependent kinetic reaction rates. At the same time, the filament configuration is actively coupled to the motor densities via the dependence of the active internal torque densities on the motor populations as well as their internal state. Appropriate ensemble averaged force-velocity relationships for the motors completes the set of equations. Numerical solutions reveal onset of dynamical instabilities via Hopf-bifurcations with oscillatory waveforms emerging from a trivial base state corresponding to a straight, non-moving flagellum.

  9. Caught Nek-ing: cilia and centrioles.

    PubMed

    Quarmby, Lynne M; Mahjoub, Moe R

    2005-11-15

    The Nek family of cell-cycle kinases is widely represented in eukaryotes and includes numerous proteins that were described only recently and remain poorly characterized. Comparing Neks in the context of clades allows us to examine the question of whether microbial eukaryotic Neks, although not strictly orthologs of their vertebrate counterparts, can provide clues to ancestral functions that might be retained in the vertebrate Neks. Relatives of the Nek2/NIMA proteins play important roles at the G2-M transition in nuclear envelope breakdown and centromere separation. Nek6, Nek7 and Nek9 also seem to regulate mitosis. By contrast, Nek1 and Nek8 have been linked with polycystic kidney disease. Results of statistical analysis indicate that the family coevolved with centrioles that function as both microtubule-organizing centers and the basal bodies of cilia. This evolutionary perspective, taken together with functional studies of microbial Neks, provides new insights into the cellular roles of the proteins and disease with which some of them have been linked. PMID:16280549

  10. Polarisation of electroweak gauge bosons at the LHC

    NASA Astrophysics Data System (ADS)

    Stirling, James; Vryonidou, Eleni

    2013-05-01

    We present results for the polarisation of gauge bosons produced at the LHC. Polarisation effects for W bosons manifest themselves in the angular distributions of the lepton and in the distributions of lepton transverse momentum and missing transverse energy. The polarisation is discussed for a range of different processes producing W bosons such as W+jets and W from top production. The relative contributions of the different polarisation states vary from process to process, reflecting the dynamics of the underlying hardscattering process. We also calculate the polarisation of the Z boson produced in association with QCD jets at the LHC.

  11. Effect of viscosity on metachrony in mucus propelling cilia.

    PubMed

    Gheber, L; Korngreen, A; Priel, Z

    1998-01-01

    In the present work we report that increasing the viscosity of the medium caused not only a decrease in the ciliary beat frequency but also changes in the metachrony and correlation between cilia. The study was performed using double and triple simultaneous photoelectric measurements on cultured ciliary cells from the frog esophagus in the viscosity range of 1-2,000 cp. We observed that increasing the viscosity intensified the fluctuations in all the measured parameters. Ciliary beat frequency decreased moderately. Even at quite high viscosities (circa 2000 cp.), cilia were still active with beating frequencies of 3-5 Hz. In addition, the degree of correlation between cilia parallel to the effective stroke direction (ESD) decreased, while that perpendicular to the ESD at a low range of viscosities remained unchanged and even increased at high viscosities. Medium viscosities in the range of 30-1,500 cp. altered the metachronal wave properties of cultured frog esophagus. The metachronal wavelength increased by up to 50%, and the wave direction changed towards more orthoplectic type of coordination. According to our recently suggested model [Gheber and Priel, 1990: Cell Motil. Cytoskeleton 16:167-181], these effects can be explained by a decrease in the temporal asymmetry of the ciliary beat. Since similar results were observed in water propelling cilia of Paramecium subjected to medium viscosity ranges of up to 40 cp. [Machemer, 1972: J. Exp. Biol. 57:239-259], we conclude that hydrodynamic interactions govern the metachronal wave properties of both mucus and water propelling cilia, though mucus propelling cilia, with their better adaptation to increased load, are affected at much higher viscosities than water propelling cilia. PMID:9453710

  12. Experimental investigation of the flow induced by artificial cilia.

    PubMed

    Hussong, J; Schorr, N; Belardi, J; Prucker, O; Rühe, J; Westerweel, J

    2011-06-21

    The fluid transport produced by rectangular shaped, magnetically actuated artificial cilia of 70 μm length and 20 μm width was determined by means of phase-locked Micro Particle Image Velocimetry (μPIV) measurements in a closed microfluidic chamber. The phase-averaged flow produced by the artificial cilia reached up to 130 μm s(-1) with an actuation cycle frequency of 10 Hz. Analysis of the measured flow data indicate that the present system is capable of achieving volume flow rates of V[combining dot above](cilia) = 14 ± 4 μl min(-1) in a micro channel of 0.5 × 5 mm(2) cross-sectional area when no back pressure is built up. This corresponds to an effective pressure gradient of 6 ± 1 Pa m(-1), which equals a pressure difference of 0.6 ± 0.1 mPa over a distance of 100 μm between two rows of cilia. These results were derived analytically from the measured velocity profile by treating the cilia as a thin boundary layer. While the cilia produce phase-averaged velocities of the order of O(10(2)μm s(-1)), time-resolved measurements showed that the flow field reverses two times during one actuation cycle inducing instantaneous velocities of up to approximately 2 mm s(-1). This shows that the flow field is dominated by fluid oscillations and flow rates are expected to increase if the beating motion of the cilia is further improved. PMID:21614349

  13. A Numerical Study of Muco-Ciliary Transport under the condition of Primary Ciliary Dyskinesia

    NASA Astrophysics Data System (ADS)

    Jayathilake, Pahala Gedara; Lee, Wan Lung; Le, Duc Vinh; Lee, Heow Pueh; Khoo, Boo Cheong

    2012-11-01

    Primary ciliary dyskinesia (PCD) is a disease due to the defects in motile cilia. A two-dimensional numerical model based on the immersed boundary method coupled with the projection method is used for a preliminary study of the flow physics of muco-ciliary transport of human respiratory tract under PCD conditions. The effects of the cilia beating amplitude, cilia beat pattern (CBP), cilia beat frequency (CBF), immotile cilia, and uncoordinated beating of cilia on mucus transport are investigated. As expected, the mucus velocity decreases as the beating amplitude and CBF decrease. The windscreen wiper motion and rigid rod motion, which are two abnormal CBPs owing to PCD, would greatly reduce the mucus transport. The mucus velocity decreases rather linearly if the number of uniformly distributed immotile cilia increases. The results further show that the mucus velocity would be slightly reduced when the uniformly distributed immotile cilia are rearranged as a cluster of immotile cilia. Furthermore, if the half of the cilia are immotile and uniformly distributed, the incoordination between motile cilia would not significantly affect the mucus velocity.

  14. Microscale flow propulsion through bioinspired and magnetically actuated artificial cilia

    PubMed Central

    Chen, Chia-Yuan; Cheng, Ling-Ying; Hsu, Chun-Chieh; Mani, Karthick

    2015-01-01

    Recent advances in microscale flow propulsion through bioinspired artificial cilia provide a promising alternative for lab-on-a-chip applications. However, the ability of actuating artificial cilia to achieve a time-dependent local flow control with high accuracy together with the elegance of full integration into the biocompatible microfluidic platforms remains remote. Driven by this motive, the current work has constructed a series of artificial cilia inside a microchannel to facilitate the time-dependent flow propulsion through artificial cilia actuation with high-speed (>40 Hz) circular beating behavior. The generated flow was quantified using micro-particle image velocimetry and particle tracking with instantaneous net flow velocity of up to 101 μm/s. Induced flow patterns caused by the tilted conical motion of artificial cilia constitutes efficient fluid propulsion at microscale. This flow phenomenon was further measured and illustrated by examining the induced flow behavior across the depth of the microchannel to provide a global view of the underlying flow propulsion mechanism. The presented analytic paradigms and substantial flow evidence present novel insights into the area of flow manipulation at microscale. PMID:26045730

  15. Microscale flow propulsion through bioinspired and magnetically actuated artificial cilia.

    PubMed

    Chen, Chia-Yuan; Cheng, Ling-Ying; Hsu, Chun-Chieh; Mani, Karthick

    2015-05-01

    Recent advances in microscale flow propulsion through bioinspired artificial cilia provide a promising alternative for lab-on-a-chip applications. However, the ability of actuating artificial cilia to achieve a time-dependent local flow control with high accuracy together with the elegance of full integration into the biocompatible microfluidic platforms remains remote. Driven by this motive, the current work has constructed a series of artificial cilia inside a microchannel to facilitate the time-dependent flow propulsion through artificial cilia actuation with high-speed (>40 Hz) circular beating behavior. The generated flow was quantified using micro-particle image velocimetry and particle tracking with instantaneous net flow velocity of up to 10(1 ) μm/s. Induced flow patterns caused by the tilted conical motion of artificial cilia constitutes efficient fluid propulsion at microscale. This flow phenomenon was further measured and illustrated by examining the induced flow behavior across the depth of the microchannel to provide a global view of the underlying flow propulsion mechanism. The presented analytic paradigms and substantial flow evidence present novel insights into the area of flow manipulation at microscale. PMID:26045730

  16. Ciliopathy-associated gene Cc2d2a promotes assembly of subdistal appendages on the mother centriole during cilia biogenesis

    PubMed Central

    Veleri, Shobi; Manjunath, Souparnika H.; Fariss, Robert N.; May-Simera, Helen; Brooks, Matthew; Foskett, Trevor A.; Gao, Chun; Longo, Teresa A.; Liu, Pinghu; Nagashima, Kunio; Rachel, Rivka A.; Li, Tiansen; Dong, Lijin; Swaroop, Anand

    2014-01-01

    The primary cilium originates from the mother centriole and participates in critical functions during organogenesis. Defects in cilia biogenesis or function lead to pleiotropic phenotypes. Mutations in centrosome-cilia gene CC2D2A result in Meckel and Joubert syndromes. Here we generate a Cc2d2a-/- mouse that recapitulates features of Meckel syndrome including embryonic lethality and multi-organ defects. Cilia are absent in Cc2d2a-/- embryonic node and other somatic tissues; disruption of cilia-dependent Shh signaling appears to underlie exencephaly in mutant embryos. The Cc2d2a-/- mouse embryonic fibroblasts (MEFs) lack cilia though mother centriole and pericentriolar proteins are detected. Odf2, associated with subdistal appendages, is absent and ninein is reduced in mutant MEFs. In Cc2d2a-/- MEFs, subdistal appendages are lacking or abnormal by transmission-EM. Consistent with this, CC2D2A localizes to subdistal appendages by immuno-EM in wild type cells. We conclude that CC2D2A is essential for the assembly of subdistal appendages, which anchor cytoplasmic microtubules and prime the mother centriole for axoneme biogenesis. PMID:24947469

  17. Modelling elliptically polarised free electron lasers

    NASA Astrophysics Data System (ADS)

    Henderson, J. R.; Campbell, L. T.; Freund, H. P.; McNeil, B. W. J.

    2016-06-01

    A model of a free electron laser (FEL) operating with an elliptically polarised undulator is presented. The equations describing the FEL interaction, including resonant harmonic radiation fields, are averaged over an undulator period and generate a generalised Bessel function scaling factor, similar to that of planar undulator FEL theory. Comparison between simulations of the averaged model with those of an unaveraged model show very good agreement in the linear regime. Two unexpected results were found. Firstly, an increased coupling to harmonics for elliptical rather than planar polarisarised undulators. Secondly, and thought to be unrelated to the undulator polarisation, a significantly different evolution between the averaged and unaveraged simulations of the harmonic radiation evolution approaching FEL saturation.

  18. Centriole distal appendages promote membrane docking, leading to cilia initiation

    PubMed Central

    Tanos, Barbara E.; Yang, Hui-Ju; Soni, Rajesh; Wang, Won-Jing; Macaluso, Frank P.; Asara, John M.; Tsou, Meng-Fu Bryan

    2013-01-01

    The distal appendages (DAPs) of centrioles have been proposed to anchor cilia to the plasma membrane, but their molecular composition, assembly, and exact function in ciliogenesis remain poorly understood. Using quantitative centrosome proteomics and superresolution microscopy, we identified five DAP components, including one previously described (CEP164), one partially characterized (CEP89 [ccdc123]), and three novel (CEP83 [ccdc41], SCLT1, and FBF1) DAP proteins. Analyses of DAP assembly revealed a hierarchy. CEP83 recruits both SCLT1 and CEP89 to centrioles. Subsequent recruitment of FBF1 and CEP164 is independent of CEP89 but mediated by SCLT1. All five DAP components are essential for ciliogenesis; loss of CEP83 specifically blocks centriole-to-membrane docking. Undocked centrioles fail to recruit TTBK2 or release CP110, the two earliest modifications found on centrioles prior to cilia assembly, revealing centriole-to-membrane docking as a temporal and spatial cue promoting cilia initiation. PMID:23348840

  19. 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

  20. 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

    2015-01-01

    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

  1. Symmetry Breaking in a Model for Nodal Cilia

    NASA Astrophysics Data System (ADS)

    Brokaw, Charles J.

    2005-03-01

    Nodal cilia are very short cilia found in the embryonic node on the ventral surface of early mammalian embryos. They create a right to left fluid flow that is responsible for determining the normal asymmetry of the internal organs of the mammalian body. To do this, the distal end of the cilium must circle in a counterclockwise sense. Computer simulations with 3-dimensional models of flagella allow examination of 3-dimensional movements such as those of nodal cilia. 3-dimensional circling motions of short cilia can be achieved with velocity controlled models, in which dynein activity is regulated by sliding velocity. If dyneins on one outer doublet are controlled by the sliding velocity experienced by that doublet, the system is symmetric, and the 3-dimensional models can show either clockwise or counterclockwise circling. My computer simulations have examined two possible symmetry breaking mechanisms: 1) dyneins on doublet N are regulated by a mixture of the sliding velocities experienced by doublets N and N+1 (numbered in a clockwise direction, looking from the base). or 2) symmetry is broken by an off-axis force that produces a right-handed twist of the axoneme, consistent with observations that some dyneins can rotate their substrate microtubules in a clockwise direction.

  2. Model Cilia - Experiments with Biomimetic Actuable Structures and Surfaces

    NASA Astrophysics Data System (ADS)

    Lloyd Carroll, R.

    2005-03-01

    The use of cilia to drive fluid flow is a common motif in living organisms, and in the tissues of higher organisms. By understanding the ways that cilia function (or do not function), potential therapies to treat human diseases (such as cystic fibrosis) may be devised. The complex hydrodynamics of flow in beating ciliary tissues (such as lung epithelial tissues) are challenging to study in cultured tissues, suggesting the need for model systems that will mimic the morphology and beat patterns of living systems. To reach this goal, we have fabricated high aspect ratio cilia-like structures with dimensions similar to those of a lung epithelial cilium (0.2 to 2.0 μm diameter by ˜6 to 10 μm long). The structures and surfaces are composed of a magneto-elastomeric nanocomposite, allowing the actuation of artificial cilia by magnetic fields. We have studied the flexibility of the materials under conditions of flow (in microfluidics channels), and will present theoretical and experimental data from various efforts at actuation. We will discuss details of the fabrication of the ciliated structures and present results of mechanical characterization. The impact of this work on the understanding of fluid flow above ciliated cells and tissues and potential applications of such model systems will also be described.

  3. Realizing the Physics of Motile Cilia Synchronization with Driven Colloids

    NASA Astrophysics Data System (ADS)

    Bruot, Nicolas; Cicuta, Pietro

    2016-03-01

    Cilia and flagella in biological systems often show large scale cooperative behaviors such as the synchronization of their beats in "metachronal waves." These are beautiful examples of emergent dynamics in biology, and are essential for life, allowing diverse processes from the motility of eukaryotic microorganisms, to nutrient transport and clearance of pathogens from mammalian airways. How these collective states arise is not fully understood, but it is clear that individual cilia interact mechanically, and that a strong and long-ranged component of the coupling is mediated by the viscous fluid. We review here the work by ourselves and others aimed at understanding the behavior of hydrodynamically coupled systems, and particularly a set of results that have been obtained both experimentally and theoretically by studying actively driven colloidal systems. In these controlled scenarios, it is possible to selectively test aspects of living motile cilia, such as the geometrical arrangement, the effects of the driving profile and the distance to no-slip boundaries. We outline and give examples of how it is possible to link model systems to observations on living systems, which can be made on microorganisms, on cell cultures or on tissue sections. This area of research has clear clinical application in the long term, as severe pathologies are associated with compromised cilia function in humans.

  4. Magnetically actuated artificial cilia for optimum mixing performance in microfluidics.

    PubMed

    Chen, Chia-Yuan; Chen, Chia-Yun; Lin, Cheng-Yi; Hu, Ya-Ting

    2013-07-21

    Contemporary lab-chip devices require efficient, high-performance mixing capability. A series of artificial cilia with embedded magnetic particles was fabricated to achieve precise flow manipulation through magnetically driven control. These fabricated structures were actuated in a homogeneous magnetic field generated by a built-in magnetic coil system for various beating cycles inside a microchannel. Three representative trajectories, namely, circular motion, back-and-forth oscillation, and a figure-of-eight pattern, of artificial cilia were designed and generated to mimic the motion of actual cilia. Homogeneous mixing of two highly viscous (>25 centipoise) dyed solutions by using the figure-of-eight trajectory achieved a mixing efficiency of approximately 86%. The underlying relationship between ciliated structures and the induced flow fields was further elucidated by performing a hydrodynamic analysis with micro-particle image velocimetry. In addition, a numerical modeling method which used a fluid structure interaction module was applied to provide quantitative 3D illustrations of induced flow patterns, including vortical structures and vortex core locations. The results reveal that both the magnitude and distribution of induced vortices primarily affect the mixing performance of two viscous flow streams. By using magnetically controlled artificial cilia along with the presented analytical paradigms, a new active flow mixing strategy was suggested to efficiently transport/agitate flows for microfluidics and biomedical applications. PMID:23685964

  5. A novel biosensor to study cAMP dynamics in cilia and flagella

    PubMed Central

    Mukherjee, Shatanik; Jansen, Vera; Jikeli, Jan F; Hamzeh, Hussein; Alvarez, Luis; Dombrowski, Marco; Balbach, Melanie; Strünker, Timo; Seifert, Reinhard; Kaupp, U Benjamin; Wachten, Dagmar

    2016-01-01

    The cellular messenger cAMP regulates multiple cellular functions, including signaling in cilia and flagella. The cAMP dynamics in these subcellular compartments are ill-defined. We introduce a novel FRET-based cAMP biosensor with nanomolar sensitivity that is out of reach for other sensors. To measure cAMP dynamics in the sperm flagellum, we generated transgenic mice and reveal that the hitherto methods determining total cAMP levels do not reflect changes in free cAMP levels. Moreover, cAMP dynamics in the midpiece and principal piece of the flagellum are distinctively different. The sole cAMP source in the flagellum is the soluble adenylate cyclase (SACY). Although bicarbonate-dependent SACY activity requires Ca2+, basal SACY activity is suppressed by Ca2+. Finally, we also applied the sensor to primary cilia. Our new cAMP biosensor features unique characteristics that allow gaining new insights into cAMP signaling and unravel the molecular mechanisms underlying ciliary function in vitro and in vivo. DOI: http://dx.doi.org/10.7554/eLife.14052.001 PMID:27003291

  6. A novel biosensor to study cAMP dynamics in cilia and flagella.

    PubMed

    Mukherjee, Shatanik; Jansen, Vera; Jikeli, Jan F; Hamzeh, Hussein; Alvarez, Luis; Dombrowski, Marco; Balbach, Melanie; Strünker, Timo; Seifert, Reinhard; Kaupp, U Benjamin; Wachten, Dagmar

    2016-01-01

    The cellular messenger cAMP regulates multiple cellular functions, including signaling in cilia and flagella. The cAMP dynamics in these subcellular compartments are ill-defined. We introduce a novel FRET-based cAMP biosensor with nanomolar sensitivity that is out of reach for other sensors. To measure cAMP dynamics in the sperm flagellum, we generated transgenic mice and reveal that the hitherto methods determining total cAMP levels do not reflect changes in free cAMP levels. Moreover, cAMP dynamics in the midpiece and principal piece of the flagellum are distinctively different. The sole cAMP source in the flagellum is the soluble adenylate cyclase (SACY). Although bicarbonate-dependent SACY activity requires Ca(2+), basal SACY activity is suppressed by Ca(2+). Finally, we also applied the sensor to primary cilia. Our new cAMP biosensor features unique characteristics that allow gaining new insights into cAMP signaling and unravel the molecular mechanisms underlying ciliary function in vitro and in vivo. PMID:27003291

  7. Primary ciliary dyskinesia and associated sensory ciliopathies

    PubMed Central

    Horani, Amjad; Ferkol, Thomas W

    2016-01-01

    Primary ciliary dyskinesia (PCD) is a genetic disease of motile cilia, which belongs to a group of disorders resulting from dysfunction of cilia, collectively known as ciliopathies. Insights into the genetics and phenotypes of PCD have grown over the last decade, in part propagated by the discovery of a number of novel cilia-related genes. These genes encode proteins that segregate into structural axonemal, regulatory, as well as cytoplasmic assembly proteins. Our understanding of primary (sensory) cilia has also expanded, and an ever-growing list of diverse conditions has been linked to defective function and signaling of the sensory cilium. Recent multicenter clinical and genetic studies have uncovered the heterogeneity of motile and sensory ciliopathies, and in some cases, the overlap between these conditions. In this review, we will describe the genetics and pathophysiology of ciliopathies in children, focusing on PCD, review emerging genotype-phenotype relationships, and diagnostic tools available for the clinician. PMID:26967669

  8. A mechanism for vertebrate Hedgehog signaling: recruitment to cilia and dissociation of SuFu–Gli protein complexes

    PubMed Central

    Tukachinsky, Hanna; Lopez, Lyle V.

    2010-01-01

    In vertebrates, Hedgehog (Hh) signaling initiated in primary cilia activates the membrane protein Smoothened (Smo) and leads to activation of Gli proteins, the transcriptional effectors of the pathway. In the absence of signaling, Gli proteins are inhibited by the cytoplasmic protein Suppressor of Fused (SuFu). It is unclear how Hh activates Gli and whether it directly regulates SuFu. We find that Hh stimulation quickly recruits endogenous SuFu–Gli complexes to cilia, suggesting a model in which Smo activates Gli by relieving inhibition by SuFu. In support of this model, we find that Hh causes rapid dissociation of the SuFu–Gli complex, thus allowing Gli to enter the nucleus and activate transcription. Activation of protein kinase A (PKA), an inhibitor of Hh signaling, blocks ciliary localization of SuFu–Gli complexes, which in turn prevents their dissociation by signaling. Our results support a simple mechanism in which Hh signals at vertebrate cilia cause dissociation of inactive SuFu–Gli complexes, a process inhibited by PKA. PMID:20956384

  9. Circularly polarised phosphorescent photoluminescence and electroluminescence of iridium complexes

    PubMed Central

    Li, Tian-Yi; Jing, Yi-Ming; Liu, Xuan; Zhao, Yue; Shi, Lin; Tang, Zhiyong; Zheng, You-Xuan; Zuo, Jing-Lin

    2015-01-01

    Nearly all the neutral iridium complexes widely used as dopants in PhOLEDs are racemic mixtures; however, this study observed that these complexes can be separated into stable optically active Λ and ∆ isomers and that their chirality is an intrinsic property. The circularly polarised phosphorescent photoluminescence (CPPPL) signals of Λ/Δ isomers are perfect mirror images with opposite polarisation and equal intensity exhibiting a “handedness” for the polarisation. For the first time, we applied the Λ/Δ iridium isomers as emitters in OLEDs, and the circularly polarised phosphorescent electroluminescence (CPPEL) spectra reveal completely positive or negative broad peaks consistent with the CPPPL spectra. The results demonstrate that the Λ/Δ isomers have potential application for 3D OLEDs because they can exhibit high efficiency and luminance, and 3D display technology based on circularly polarised light is the most comfortable for the eyes. PMID:26446521

  10. The Primary Cilium: A Signaling Center During Vertebrate Development

    PubMed Central

    Goetz, Sarah C.

    2011-01-01

    The primary cilium has recently stepped into the spotlight, as a flood of data demonstrate that this organelle has crucial roles in vertebrate development and human genetic diseases. Cilia are required for the response to developmental signals, and evidence is accumulating that the primary cilium is specialized for Hedgehog (Hh) signal transduction. Formation of cilia, in turn, is regulated by other signaling pathways, possibly including the planar cell polarity pathway. The cilium therefore represents a nexus for signaling pathways during development. The connections between cilia and developmental signaling have begun to clarify the basis of human diseases associated with ciliary dysfunction. PMID:20395968

  11. Cilia-based flow network in the brain ventricles.

    PubMed

    Faubel, Regina; Westendorf, Christian; Bodenschatz, Eberhard; Eichele, Gregor

    2016-07-01

    Cerebrospinal fluid conveys many physiologically important signaling factors through the ventricular cavities of the brain. We investigated the transport of cerebrospinal fluid in the third ventricle of the mouse brain and discovered a highly organized pattern of cilia modules, which collectively give rise to a network of fluid flows that allows for precise transport within this ventricle. We also discovered a cilia-based switch that reliably and periodically alters the flow pattern so as to create a dynamic subdivision that may control substance distribution in the third ventricle. Complex flow patterns were also present in the third ventricles of rats and pigs. Our work suggests that ciliated epithelia can generate and maintain complex, spatiotemporally regulated flow networks. PMID:27387952

  12. IFT46 plays crucial roles in craniofacial and cilia development.

    PubMed

    Park, Inji; Lee, Hyun-Kyung; Kim, Chowon; Ismail, Tayaba; Kim, Yoo-Kyung; Park, Jeen-Woo; Kwon, Oh-Shin; Kang, Beom Sik; Lee, Dong-Seok; Park, Tae-Joo; Park, Mae-Ja; Choi, Sun-Cheol; Lee, Hyun-Shik

    2016-08-26

    The intraflagellar transport (IFT) system is essential for bidirectional movement of ciliary components from the basal body to the tip beneath the ciliary sheath and is conserved for cilia and flagella formation in most vertebrates. IFT complex A is involved in anterograde trafficking, whereas complex B is involved in retrograde trafficking. IFT46 is well known as a crucial component of IFT complex B, however, its developmental functions are poorly understood. In this study, we investigated the novel functions of IFT46 during vertebrate development, especially, ciliogenesis and neurogenesis, because IFT46 is strongly expressed in both multiciliated cells of epithelial and neural tissues. Knockdown of IFT46 using morpholino microinjections caused shortening of the body axis as well as the formation of fewer and shorter cilia. Furthermore, loss of IFT46 down-regulated the expression of the neural plate and neural tube markers, thus may influence Wnt/planar cell polarity and the sonic hedgehog signaling pathway during neurogenesis. In addition, loss of IFT46 caused craniofacial defects by interfering with cartilage formation. In conclusion, our results depict that IFT46 plays important roles in cilia as well as in neural and craniofacial development. PMID:27320864

  13. 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.

  14. Nanoscale Fluidics: Using magnetic nanorods as model cilia

    NASA Astrophysics Data System (ADS)

    Hao, Jing; Ben, Wilde; Jeremy, Cribb; Chris, Dwyer; Jay, Fisher; Kalpit, Desai; Leandra, Vicci; Russell, M. Taylor, II; Richard, Superfine

    2003-11-01

    The beating of cilia and flagella, slender cylinders 250 nanometers in diameter with lengths from 7 to 50 microns, is ubiquitous in biology. The fluid dynamics produced by the cilia or flagella motion is responsible for organism feeding, propulsion, for bacterial clearance in the lungs and for the right-left asymmetry in vertebrates. We are developing a model system for cilia beating through the use of magnetic nanorods. Using anodized aluminum oxide (AAO) membranes as templates, magnetic rods of permalloy with a diameter of 100 and 200 nm have been fabricated. We will describe the details of fabrication and characterization, and discuss methods used to study the hydrodynamic behavior of these nanorods in aqueous solutions under applied magnetic fields. Movies of these nanorods in oscillating 3-D magnetic fields generated by our 3-dimensional force microscopy (3DFM) clearly show bead motion in vortices around the nanorod. Deliberately transporting beads near the rods by laser trap, we can reproducibly study the hydrodynamic behavior around the nanorods and the influence of local rheological properties.

  15. Robust estimation of the motile cilia beating frequency.

    PubMed

    Meste, O; Brau, F; Guyon, A

    2015-10-01

    The estimation of the cilia beating frequency (CBF) is of great interest in understanding how the CBF modulates liquid fluxes and how it is controlled by the ciliated cell intra- and/or extracellular medium composition in physiological processes. Motion artifacts and camera defaults may hinder the computation of the frequency variations during long-lasting experiments. We have developed a new analysis approach consisting of a preliminary corrective step (removal of a grid pattern on the image sequence and shift compensation), followed by a harmonic model of the observed cilia using a maximum likelihood estimator framework. It is shown that a more accurate estimation of the frequency can be obtained by averaging the squared Fourier transform of individual pixels followed by a particular summation over the different frequencies, namely the compressed spectrum. Robustness of the proposed method over traditional approaches is shown by several examples and simulations. The method is then applied to images of samples containing ciliated ependymal cells located in the third cerebral ventricle of mouse brains, showing that even small variations in CBF in response to changes in the amount of oxygenation, pH or glucose were clearly visible in the computed frequencies. As a conclusion, this method reveals a fine metabolic tuning of the cilia beating in ependimocytes lining the third cerebral ventricle. Such regulations are likely to participate in homeostatic mechanisms regulating CSF movements and brain energy supply. PMID:26215519

  16. Tubulin glycylases and glutamylases have distinct functions in stabilization and motility of ependymal cilia

    PubMed Central

    Bosch Grau, Montserrat; Gonzalez Curto, Gloria; Rocha, Cecilia; Magiera, Maria M.; Marques Sousa, Patricia; Giordano, Tiziana

    2013-01-01

    Microtubules are subject to a variety of posttranslational modifications that potentially regulate cytoskeletal functions. Two modifications, glutamylation and glycylation, are highly enriched in the axonemes of most eukaryotes, and might therefore play particularly important roles in cilia and flagella. Here we systematically analyze the dynamics of glutamylation and glycylation in developing mouse ependymal cilia and the expression of the corresponding enzymes in the brain. By systematically screening enzymes of the TTLL family for specific functions in ependymal cilia, we demonstrate that the glycylating enzymes TTLL3 and TTLL8 were required for stability and maintenance of ependymal cilia, whereas the polyglutamylase TTLL6 was necessary for coordinated beating behavior. Our work provides evidence for a functional separation of glutamylating and glycylating enzymes in mammalian ependymal cilia. It further advances the elucidation of the functions of tubulin posttranslational modifications in motile cilia of the mammalian brain and their potential importance in brain development and disease. PMID:23897886

  17. On the compatibility of polarisable and non-polarisable models for liquid water

    NASA Astrophysics Data System (ADS)

    Bachmann, Stephan Jan; van Gunsteren, Wilfred F.

    2014-11-01

    The properties of water at physiological conditions can be modelled at different levels of resolution: (1) sub-atomic models that take into account electronic degrees of freedom, (2) atomic models that only account for atomic degrees of freedom and (3) supra-molecular models that only involve some supra-molecular degrees of freedom. To enhance the computational efficiency of molecular simulation, models at different levels of resolution should be simultaneously usable for different parts of a system for which the level of detail of interest is different. This requires these different types of models to be compatible with each other. In the present study, the compatibility of two polarisable models for liquid water, COS/G2 and COS/D, with a non-polarisable model for liquid water, SPC, is investigated. It is shown that these models are compatible. The polarisable models can thus be used to solvate biomolecules described by a biomolecular force field that is compatible with the SPC water model.

  18. The Strange Quark Polarisation from COMPASS data

    SciTech Connect

    Kouznetsov, O.

    2009-12-17

    The strange quark helicity distribution {delta}s(x) was derived at LO from the inclusive asymmetry A{sub a,d} and the semi-inclusive asymmetries A{sub 1,d}{sup {pi}}{sup +}, A{sub 1,d}{sup {pi}}{sup -}, A{sub 1,d}{sup K+}, A{sub 1,d}{sup K-}, measured by COMPASS in polarised deep inelastic muon-deuteron scattering. The distribution of {delta}s(x) is compatible with zero in the whole measured range. The value of the first moment of {delta}s and its error are very sensitive to the assumed value of the ratio of the s-bar-quark to u-quark fragmentation functions into positive kaons {integral}D(K+/s)(z)dz/{integral}D{sub u}{sup K+}(z)dz.

  19. Null point of discrimination in crustacean polarisation vision.

    PubMed

    How, Martin J; Christy, John; Roberts, Nicholas W; Marshall, N Justin

    2014-07-15

    The polarisation of light is used by many species of cephalopods and crustaceans to discriminate objects or to communicate. Most visual systems with this ability, such as that of the fiddler crab, include receptors with photopigments that are oriented horizontally and vertically relative to the outside world. Photoreceptors in such an orthogonal array are maximally sensitive to polarised light with the same fixed e-vector orientation. Using opponent neural connections, this two-channel system may produce a single value of polarisation contrast and, consequently, it may suffer from null points of discrimination. Stomatopod crustaceans use a different system for polarisation vision, comprising at least four types of polarisation-sensitive photoreceptor arranged at 0, 45, 90 and 135 deg relative to each other, in conjunction with extensive rotational eye movements. This anatomical arrangement should not suffer from equivalent null points of discrimination. To test whether these two systems were vulnerable to null points, we presented the fiddler crab Uca heteropleura and the stomatopod Haptosquilla trispinosa with polarised looming stimuli on a modified LCD monitor. The fiddler crab was less sensitive to differences in the degree of polarised light when the e-vector was at -45 deg than when the e-vector was horizontal. In comparison, stomatopods showed no difference in sensitivity between the two stimulus types. The results suggest that fiddler crabs suffer from a null point of sensitivity, while stomatopods do not. PMID:24737768

  20. The strange history of polarised neutrons in Australia

    NASA Astrophysics Data System (ADS)

    Hicks, T. J.

    2016-04-01

    The history of polarised neutrons in Australia is unusual firstly because of the particular access that individuals in universities had to the facilities at the reactor site and because this resulted in the experiments being done almost all being with polarisation analysis. Two instruments were initially available. One was a conventional instrument albeit with a tilting counter. The other was a primitive polarisation analysis instrument purpose built for diffuse scattering. This latter instrument evolved over more than thirty years and produced results ranging from the separation of magnetic and nuclear diffuse scattering, for which it was conceived, to the isolation of magnetic features in inelastic spectra.

  1. A facile template-free approach to magnetodriven, multifunctional artificial cilia.

    PubMed

    Timonen, Jaakko V I; Johans, Christoffer; Kontturi, Kyösti; Walther, Andreas; Ikkala, Olli; Ras, Robin H A

    2010-08-01

    Flexible and magnetic artificial cilia were grown on various substrates by a facile bottom-up approach based on template-free magnetic assembly. The magnetic cilia formed spontaneously from a suspension of micrometer-sized ferromagnetic particles and elastomeric polymer in a liquid solvent when dried in an external magnetic field. The cilia mimics were mechanically stable even in the absence of an external magnetic field and a solvent due to the polymer, which acted as "glue" holding the particles together and connecting the cilia to the substrate. The length of the magnetic cilia was in the millimeter range, that is, two to three orders of magnitude times the length of typical biological cilia. The aspect ratio reached values over 100 and was tunable with the magnetic field gradient and the size of the ferromagnetic particles. The cilia mimics responded to an external magnetic field by reversibly bending along the field. The bending actuation was sufficiently powerful to allow two functions: to translate macroscopic nonmagnetic objects placed over the cilia mimics and to mix liquids of even high viscosity. The mechanical properties of the magnetic cilia could be easily tuned by changing the impregnating polymer. The particularly simple template-free construction and fixation on various surfaces suggest applications as an externally controllable surface. PMID:20695442

  2. The zebrafish fleer gene encodes an essential regulator of cilia tubulin polyglutamylation.

    PubMed

    Pathak, Narendra; Obara, Tomoko; Mangos, Steve; Liu, Yan; Drummond, Iain A

    2007-11-01

    Cilia and basal bodies are essential organelles for a broad spectrum of functions, including the development of left-right asymmetry, kidney function, cerebrospinal fluid transport, generation of photoreceptor outer segments, and hedgehog signaling. Zebrafish fleer (flr) mutants exhibit kidney cysts, randomized left-right asymmetry, hydrocephalus, and rod outer segment defects, suggesting a pleiotropic defect in ciliogenesis. Positional cloning flr identified a tetratricopeptide repeat protein homologous to the Caenorhabditis elegans protein DYF1 that was highly expressed in ciliated cells. flr pronephric cilia were shortened and showed a reduced beat amplitude, and olfactory cilia were absent in mutants. flr cilia exhibited ultrastructural defects in microtubule B-tubules, similar to axonemes that lack tubulin posttranslational modifications (polyglutamylation or polyglycylation). flr cilia showed a dramatic reduction in cilia polyglutamylated tubulin, indicating that flr encodes a novel modulator of tubulin polyglutamylation. We also found that the C. elegans flr homologue, dyf-1, is also required for tubulin polyglutamylation in sensory neuron cilia. Knockdown of zebrafish Ttll6, a tubulin polyglutamylase, specifically eliminated tubulin polyglutamylation and cilia formation in olfactory placodes, similar to flr mutants. These results are the first in vivo evidence that tubulin polyglutamylation is required for vertebrate cilia motility and structure, and, when compromised, results in failed ciliogenesis. PMID:17761526

  3. Exploiting cellophane birefringence to generate radially and azimuthally polarised vector beams

    NASA Astrophysics Data System (ADS)

    Kalwe, Johnston; Neugebauer, Martin; Ominde, Calvine; Leuchs, Gerd; Rurimo, Geoffrey; Banzer, Peter

    2015-03-01

    We exploit the birefringence of cellophane to convert a linearly polarised Gaussian beam into either a radially or azimuthally polarised beam. For that, we fabricated a low-cost polarisation mask consisting of four segments of cellophane. The fast axis of each segment is oriented appropriately in order to rotate the polarisation of the incident linearly polarised beam as desired. To ensure the correct operation of the polarisation mask, we tested the polarisation state of the generated beam by measuring the spatial distribution of the Stokes parameters. Such a device is very cost efficient and allows for the generation of cylindrical vector beams of high quality.

  4. Biochemical studies of olfaction: isolation, characterization, and odorant binding activity of cilia from rainbow trout olfactory rosettes.

    PubMed

    Rhein, L D; Cagan, R H

    1980-08-01

    The role of cilia in recognition of olfactory stimuli has been controversial. Cilia from the intact olfactory rosettes of the rainbow trout Salmo gairdneri were isolated, characterized biochemically, and examined by electron microscopy. The markers studied are those associated with cilia in other organisms. Dynein arms contain Mg2+-AtPase; this enzyme was enriched in the isolated cilia preparation. Guanine nucleotides are associated with the outer microtubule doublets of cilia but adenine nucleotides are not; a substantial enrichment in guanine, relative to adenine, was found in the cilia preparation. Tubulin, the structural protein component of microtubules, occurs in large amounts in cilia. Disc gel electrophoresis indicated tubulin in the cilia preparation. Electron microscopy confirmed the presence of cilia in the isolated preparation. Rainbow trout have an acute sense of smell and many amino acids are odorants to this species. Functional activity of the cilia preparation relevant to odorant recognition was assessed by using binding of radioactively labeled odorant amino acids. L-Alanine, L-serine, L-threonine, L-lysine, and D-alanine bound to the cilia preparation. This study provides direct biochemical evidence that olfactory cilia bind odorant molecules and supports the hypothesis that odorant recognition sites are integral parts of the cilia. PMID:6449006

  5. Polarised stereo endoscope and narrowband detection for minimal access surgery

    PubMed Central

    Clancy, Neil T.; Arya, Shobhit; Qi, Ji; Stoyanov, Danail; Hanna, George B.; Elson, Daniel S.

    2014-01-01

    Polarisation imaging has the potential to provide enhanced contrast based on variations in the optical properties, such as scattering or birefringence, of the tissue of interest. Examining the signal at different wavebands in the visible spectrum also allows interrogation of different depths and structures. A stereo endoscope has been adapted to allow snapshot acquisition of orthogonal linear polarisation images to generate difference of linear polarisation images. These images are acquired in three narrow bands using a triple-bandpass filter and pair of colour cameras. The first in vivo results, acquired during a surgical procedure on a porcine subject, are presented that show wavelength dependent variations in vessel visibility and an increase in contrast under polarised detection. PMID:25574424

  6. Thermodynamics of continuous media with permanent electric polarisation and magnetisation

    NASA Astrophysics Data System (ADS)

    Brechet, Sylvain; Ansermet, Jean-Philippe

    2012-11-01

    The thermodynamics of an electrically charged, multicomponent fluid with permanent electric polarisation, permanent magnetisation and intrinsic vorticity is analysed in the presence of electromagnetic fields with magnetoelectric coupling in the classical limit. Three equations characterising the fluid are derived: a thermostatic equilibrium equation, a reversible and an irreversible thermodynamic evolution equation. These equations are obtained by taking into account the first and second laws of thermodynamics, the chemical reactions, the second law of Newton in translation and in rotation, the local time evolution of the permanent polarisation and the permanent magnetisation, and Maxwell's equations. Explicit expressions for the temperature and the chemical potentials are derived in terms of the electromagnetic fields, the permanent electric polarisation, the permanent magnetisation, the intrinsic vorticity and the magnetoelectric coupling. The analysis of the irreversible thermodynamics yields novel dissipative equations accounting in particular for dielectrophoresis, magnetophoresis, the relaxation of the permanent electric polarisation and the permanent magnetisation, and other properties of electrorheological and magnetorheological fluids.

  7. Integrins are required for cardioblast polarisation in Drosophila

    PubMed Central

    2012-01-01

    Background The formation of a tubular organ, such as the heart, requires the communication of positional and polarity signals between migratory cells. Key to this process is the establishment of a new luminal domain on the cell surface, generally from the apical domain of a migratory cell. This domain will also acquire basal properties, as it will produce a luminal extracellular matrix. Integrin receptors are the primary means of cell adhesion and adhesion signaling with the extracellular matrix. Here we characterise the requirement of Integrins in a genetic model of vasculogenesis, the formation of the heart in Drosophila. Results As with vertebrates, the Drosophila heart arises from lateral mesoderm that migrates medially to meet their contralateral partners, to then assemble a midline vessel. During migration, Integrins are among the first proteins restricted to the presumptive luminal domain of cardioblasts. Integrins are required for normal levels of leading edge membrane motility. Apical accumulation of Integrins is enhanced by Robo, and reciprocally, apicalisation of luminal factors like Slit and Robo requires Integrin function. Integrins may provide a template for the formation of a lumen by stabilising lumen factors like Robo. Subsequent to migration, Integrin is required for normal cardioblast alignment and lumen formation. This phenotype is most readily modified by other mutations that affect adhesion, such as Talin and extracellular matrix ligands. Conclusion Our findings reveal an instructive role for Integrins in communicating polarising information to cells during migration, and during transition to an epithelial tube structure. PMID:22353787

  8. Polarised nuclei for neutron science: recent applications and perspectives

    NASA Astrophysics Data System (ADS)

    Glättli, Hans

    2004-08-01

    Neutron scattering on nuclei is spin dependent, particularly strongly for 1H. The means to achieve large nuclear polarisations and its use for structure analysis or as spin-handling device are reviewed. High resolution (diffraction) as well as low resolution (SANS) measurements can benefit from polarised nuclei by changing selectively the form factors of Bragg reflections or the contrasts (the scattering length density profiles) in SANS. The internal structure of ribosomes and the conformation of polymers in solution have been investigated by this method. A numerical simulation is presented to show the influence of steady-state polarisation of protons on the scattering from a protein-ARN model complex. In addition, a more recent technique, time-resolved SANS is described. It makes use of spatial polarisation gradients created around paramagnetic centres at the onset of nuclear polarisation. Such polarisation domains can enhance considerably the scattering amplitude of free radicals and thus contribute to determine their positions inside a complex protein. Examples of possible future experiments are proposed which combine simultaneously the selectivity of solid-state NMR techniques and neutron scattering.

  9. The sunstone and polarised skylight: ancient Viking navigational tools?

    NASA Astrophysics Data System (ADS)

    Ropars, Guy; Lakshminarayanan, Vasudevan; Le Floch, Albert

    2014-10-01

    Although the polarisation of the light was discovered at the beginning of the nineteenth century, the Vikings could have used the polarised light around the tenth century in their navigation to America, using a 'sunstone' evoked in the Icelandic Sagas. Indeed, the birefringence of the Iceland spar (calcite), a common crystal in Scandinavia, permits a simple observation of the axis of polarisation of the skylight at the zenith. From this, it is possible to guess the azimuth of a hidden Sun below the horizon, for instance. The high sensitivity of the differential method provided by the ordinary and extraordinary beams of calcite at its so-called isotropy point is about two orders higher than that of the best dichroic polariser and permits to reach an accuracy of ±1° for the Sun azimuth (at sunrise and sunset). Unfortunately, due to the relative fragility of calcite, only the so-called Alderney crystal was discovered on board a 16th ancient ship. Curiously, beyond its use as a sunstone by the Vikings, during these last millennia calcite has led to the discovery of the polarisation of the light itself by Malus and is currently being used to detect the atmospheres of exoplanets. Moreover, the differential method for the light polarisation detection is widely used in the animal world.

  10. Optically induced dynamic nuclear spin polarisation in diamond

    NASA Astrophysics Data System (ADS)

    Scheuer, Jochen; Schwartz, Ilai; Chen, Qiong; Schulze-Sünninghausen, David; Carl, Patrick; Höfer, Peter; Retzker, Alexander; Sumiya, Hitoshi; Isoya, Junichi; Luy, Burkhard; Plenio, Martin B.; Naydenov, Boris; Jelezko, Fedor

    2016-01-01

    The sensitivity of magnetic resonance imaging (MRI) depends strongly on nuclear spin polarisation and, motivated by this observation, dynamical nuclear spin polarisation has recently been applied to enhance MRI protocols (Kurhanewicz et al 2011 Neoplasia 13 81). Nuclear spins associated with the 13C carbon isotope (nuclear spin I = 1/2) in diamond possess uniquely long spin lattice relaxation times (Reynhardt and High 2011 Prog. Nucl. Magn. Reson. Spectrosc. 38 37). If they are present in diamond nanocrystals, especially when strongly polarised, they form a promising contrast agent for MRI. Current schemes for achieving nuclear polarisation, however, require cryogenic temperatures. Here we demonstrate an efficient scheme that realises optically induced 13C nuclear spin hyperpolarisation in diamond at room temperature and low ambient magnetic field. Optical pumping of a nitrogen-vacancy centre creates a continuously renewable electron spin polarisation which can be transferred to surrounding 13C nuclear spins. Importantly for future applications we also realise polarisation protocols that are robust against an unknown misalignment between magnetic field and crystal axis.