Comparative genomics: prediction of the ciliary and basal body proteome

Defects in mammalian cilia lead to a range of diseases, but our understanding of the composition of these organelles and of the basal bodies from which they arise is limited. Two recent studies used comparative genomics to predict the ciliary and basal body proteomes, providing datasets that are rich sources of human disease gene candidates.     

Patterns of basal body addition in ciliary rows in Tetrahymena

Most naked basal bodies visualized in protargol stains on the surface of Tetrahymena are new basal bodies which have not yet developed cilia. The rarity of short cilia is explained by the rapid development of the ciliary shaft once it begins to grow. The high frequency of naked basal bodies (about 50 percent) in log cultures indicates that the interval between assembly of the basal body and the initiation of the cilium is long, approximately a full cell cycle. Naked basal bodies are more frequent in the mid and posterior parts of the cell and two or more naked basal bodies may be associated with one ciliated basal body in these regions. Daughter cells produced at division are apparently asymmetric with respect to their endowment of new and old organelles.     

Drosophila transition fibers are essential for IFT-dependent ciliary elongation but not basal body docking and ciliary budding

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Cytochalasin D inhibits basal body migration and ciliary elongation in quail oviduct epithelium

Summary The effects of cytochalasin D (CD) were studied by scanning (SEM) and transmission (TEM) electron-microscopic examination at different stages of ciliary differentiation in epithelial cells of quail oviduct. Immature quails were prestimulated by estradiol benzoate injections to induce ciliogenesis in the undifferentiated oviduct. After 24 h of CD culture, SEM study revealed inhibition of ciliogenesis and dilation of the apex of non-ciliated cells. TEM study showed that 2 h of CD treatment produced dilation of lateral intercellular spaces, after 6 h of treatment, this resulted in intracellular macrovacuolation. Vacuoles were surrounded by aggregates of dense felt-like material. CD also induced the disappearance of microvilli, and rounding of the apical surface of undifferentiated cells and those blocked in ciliogenesis. Centriologenesis was not inhibited by CD; basal bodies assembled in generative complexes in the supranuclear region after 24 h of treatment. However, the migration of mature basal bodies towards the apical surface was impaired. Instead, they anchored onto the membrane of intracellular vacuoles; growth of cilia was induced in the vacuole lumen. Cilium elongation was disturbed, giving abnormally short cilia with a dilated tip; microtubules failed to organize correctly.     

Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation

Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.     

Elucidation of basal body and centriole functions in Chlamydomonas reinhardtii

In eukaryotic cells, basal bodies and their structural equivalents, centrioles, play essential roles. They are needed for the assembly of flagella or cilia as well as for cell division. Chlamydomonas reinhardtii provides an excellent model organism for the study of the basal body and centrioles. Genes for two new members of the tubulin superfamily are needed for basal body/centriole duplication. In addition, other genes that play roles in the duplication and segregation of basal bodies are discussed.     

Regulation of anterior chamber drainage by bicarbonate-sensitive soluble adenylyl cyclase in the ciliary body

Glaucoma is a leading cause of blindness affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure (IOP). IOP results from the resistance to drainage of aqueous humor (AH) produced by the ciliary body in a process requiring bicarbonate. Once secreted into the anterior chamber, AH drains from the eye via two pathways: uveoscleral and pressure-dependent or conventional outflow (C(t)). Modulation of "inflow" and "outflow" pathways is thought to occur via distinct, local mechanisms. Mice deficient in the bicarbonate channel bestrophin-2 (Best2), however, exhibit a lower IOP despite an increase in AH production. Best2 is expressed uniquely in nonpigmented ciliary epithelial (NPE) cells providing evidence for a bicarbonate-dependent communicative pathway linking inflow and outflow. Here, we show that bicarbonate-sensitive soluble adenylyl cyclase (sAC) is highly expressed in the ciliary body in NPE cells, but appears to be absent from drainage tissues. Pharmacologic inhibition of sAC in mice causes a significant increase in IOP due to a decrease in C(t) with no effect on inflow. In mice deficient in sAC IOP is elevated, and C(t) is decreased relative to wild-type mice. Pharmacologic inhibition of sAC did not alter IOP or C(t) in sAC-deficient mice. Based on these data we propose that the ciliary body can regulate C(t) and that sAC serves as a critical sensor of bicarbonate in the ciliary body regulating the secretion of substances into the AH that govern outflow facility independent of pressure.     

The two domains of centrin have distinct basal body functions in Tetrahymena

The basal body is a microtubule-organizing center responsible for organizing the cilium, a structure important for cell locomotion and sensing of the surrounding environment. A widely conserved basal body component is the Ca(2+)-binding protein centrin. Analyses of centrin function suggest a role in basal body assembly and stability; however, its molecular mechanisms remain unclear. Here we describe a mutagenic strategy to study the function and essential nature of the various structural features of Cen1 in the ciliate Tetrahymena. We find that the two domains of Cen1 are both essential, and examination of strains containing mutant CEN1 alleles indicates that there are two predominant basal body phenotypes: misorientation of newly assembled basal bodies and stability defects. The results also show that the two domains of Cen1 are able to bind Ca(2+) and that perturbation of Ca(2+) binding affects Cen1 function. In all, the data suggest that the two domains of Cen1 have distinct functions.     

Regulation of outer-arm-dynein activity by phosphorylation and control of ciliary beat frequency

Summary Ciliary beating is empowered by a mechanochemical enzyme, dynein, which appears as two rows of projections on doublet microtubules. While inner-arm dyneins modulate beat form, outer-arm dynein empowers ciliary beat and sets beat frequency. Beat frequency is controlled via phosphorylation of outer-arm dynein. UsingParamecium tetraurelia as model system, we have previously identified a regulatory light chain of outer-arm dynein (22S dynein), M_r29 (p29), whose phosphorylation is cAMP-dependent. The phosphorylation state of the p29 in 22 S dynein determines in vitro microtubule translocation velocity. Although in vitro phosphorylation of p29 takes place in a short time, the percent change ist significantly less than the percent change in dynein activation, or in ciliary beat frequency. A potential mechanism that explains how a few activated dyneins can change ciliary beating is discussed.     

Regulation of T-cell functions by L-lactate

Lactate is a product of glycolytically active macrophages. After stimulation with concanavalin A accessory cell-depleted splenic T-cell populations were found to produce only minute amounts of T-cell growth factor (TCGF); but substantial amounts of TCGF were produced if the cultures were supplemented either with splenic adherent cells or with lactate but not with interleukin-1 (IL-1). IL-1 was capable, however, of supporting TCGF production by the thymoma subline EL4-6.1. TCGF production in cultures of accessory cell-depleted splenic T-cell populations was demonstrable with 10(-3) M L-lactate, and optimal responses (plateau level) were obtained with 4-6 X 10(-2) M L-lactate. Cultures of macrophages were found to accumulate up to 5 X 10(-2) M lactate. Our experiments indicate, therefore, that lactate serves as a regulatory signal by which macrophage-like accessory cells enhance helper-T-cell functions. Lactate is apparently not the only mediator of accessory cell function since plateau levels of TCGF production were markedly lower with lactate than with splenic accessory cells; but L-lactate was found also to determine the magnitude of T-cell-mediated immune responses in vivo and in cultures of unfractionated lymphocyte populations. The production of interferon in accessory cell-depleted and concanavalin A-treated T-cell cultures, however, was not significantly affected by lactate. Concanavalin A-stimulated splenic T-cell populations were found to consume glucose rapidly and to release lactate into the supernatant. This indicates that the cells contain more lactate and pyruvate than they can utilize by their respiratory metabolism. The administration of external lactate or pyruvate was found to inhibit the utilization of glucose by the mitogenically stimulated T cells.     

Regulation of integrin functions by N-glycans

Integrins are cell surface transmembrane glycoproteins that function as adhesion receptors in cell-ECM interactions and link matrix proteins to the cytoskeleton. Integrins play an important role in cytoskeleton organization and in the transduction of intracellular signals, regulating various processes such as proliferation, differentiation, apoptosis, and cell migration. Although integrin-mediated adhesion is based on the binding of alpha and beta subunits to a defined peptide sequence, the strength of this binding is modulated by various factors including the status of glycosylation of integrin. Glycosylation reactions are catalyzed by the catalytic action of glycosyltransferases, such as N-acetylglucosaminyltransferase III, V and alpha1, 6 fucosyltransferase, etc., which catalyze the formation of glycosidic bonds. This review summarizes effects of the posttranslational modification of N-glycans of alpha3beta1 and alpha5beta1 integrins on their association, activation and biological functions, by using biochemical and genetic approaches.     

MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis

Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and related ciliopathies present with overlapping phenotypes and display considerable allelism between at least twelve different genes of largely unexplained function. We demonstrate that the conserved C. elegans B9 domain (MKS-1, MKSR-1, and MKSR-2), MKS-3/TMEM67, MKS-5/RPGRIP1L, MKS-6/CC2D2A, NPHP-1, and NPHP-4 proteins exhibit essential, collective functions at the transition zone (TZ), an underappreciated region at the base of all cilia characterized by Y-shaped assemblages that link axoneme microtubules to surrounding membrane. These TZ proteins functionally interact as members of two distinct modules, which together contribute to an early ciliogenic event. Specifically, MKS/MKSR/NPHP proteins establish basal body/TZ membrane attachments before or coinciding with intraflagellar transport-dependent axoneme extension and subsequently restrict accumulation of nonciliary components within the ciliary compartment. Together, our findings uncover a unified role for eight TZ-localized proteins in basal body anchoring and establishing a ciliary gate during ciliogenesis, and suggest that disrupting ciliary gate function contributes to phenotypic features of the MKS/NPHP disease spectrum.     

New Tetrahymena basal body protein components identify basal body domain structure

Basal bodies organize the nine doublet microtubules found in cilia. Cilia are required for a variety of cellular functions, including motility and sensing stimuli. Understanding this biochemically complex organelle requires an inventory of the molecular components and the contribution each makes to the overall structure. We define a basal body proteome and determine the specific localization of basal body components in the ciliated protozoan Tetrahymena thermophila. Using a biochemical, bioinformatic, and genetic approach, we identify 97 known and candidate basal body proteins. 24 novel T. thermophila basal body proteins were identified, 19 of which were localized to the ultrastructural level, as seen by immunoelectron microscopy. Importantly, we find proteins from several structural domains within the basal body, allowing us to reveal how each component contributes to the overall organization. Thus, we present a high resolution localization map of basal body structure highlighting important new components for future functional studies.     

Coordinated ciliary beating requires Odf2-mediated polarization of basal bodies via basal feet

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Highlights? Mice expressing truncated Odf2 cough and sneeze due to primary ciliary dyskinesia ? Full-length Odf2 is needed for the formation of basal body-associated basal feet ? In the absence of basal feet, basal bodies fail to align with planar polarity cues ? Polarization of basal bodies by Odf2 is required for coordinated ciliary beating     

Regulation of ciliary beat frequency by autonomic mechanisms: in vitro

The ciliated epithelium of the mammalian trachea separates the neurohumoral milieu of the tissue from that of the environment of the airway lumen. To determine whether specific autonomic receptors regulating ciliary beat frequency (CBF) were located on mucosal or serosal sides, we measured CBF by heterodyne mode correlation analysis laser light scattering in bovine tracheal tissues mounted in a two-sided chamber. A beta 2-adrenergic agonist, fenoterol, at 10(-7) M, stimulated serosal CBF from 7.9 +/- 1.3 to 20.2 +/- 5.8 Hz (P less than 0.01) and mucosal CBF from 6.6 +/- 0.9 to 14.7 +/- 4.6 Hz (P less than 0.01). A muscarinic cholinergic agonist, methacholine, at 10(-7) M, increased mucosal CBF from 8.4 +/- 1.0 to 19.5 +/- 5.5 Hz (P less than 0.01) and serosal CBF from 8.0 +/- 0.9 to 15.4 +/- 5.0 Hz (P less than 0.01). The differences in stimulation of CBF on the mucosal and serosal sides between fenoterol and methacholine were significant (P less than 0.01). Studies in which these autonomic agonist stimulating effects were inhibited by their respective antagonists, propranolol and atropine sulfate, demonstrated that CBF can be regulated independently by mediators both in the submucosa and within the mucus lining.     

Regulation of ciliary pattern in ciliates.

The modes of pattern regulation found in the ciliates Dileptus and Paraurostyla are compared. These forms are systematically distant but both possess very extensive regenerative capacities. They are characterized by 2 types of ciliary patterns: the ciliature of Dileptus has largely a simple pattern composed of single kinetosomes while that of Paraurostyla has a complex pattern composed of aggregates of kinetosomes interconnected by amorphic matter. In both ciliates a change in cell size evokes pattern regulation which differs substantially in the extent of pattern replacement, timing, and localization of morphogenetic activities. It is concluded that these differences result from the pattern constituents.