Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons.

Odorant signal transduction occurs in the specialized cilia of the olfactory sensory neurons. Considerable biochemical evidence now indicates that this process could be mediated by a G protein-coupled cascade using cyclic AMP as an intracellular second messenger. A stimulatory G protein alpha subunit is expressed at high levels in olfactory neurons and is specifically enriched in the cilia, as is a novel form of adenylyl cyclase. This implies that the olfactory transduction cascade might involve unique molecular components. Electrophysiological studies have identified a cyclic nucleotide-activated ion channel in olfactory cilia. These observations provide evidence for a model in which odorants increase intracellular cAMP concentration, which in turn activates this channel and depolarizes the sensory neuron. An analogous cascade regulating a cGMP-gated channel mediates visual transduction in photoreceptor cells. The formal similarities between olfactory and visual transduction suggest that the two systems might use homologous channels. Here we report the molecular cloning, functional expression and characterization of a channel that is likely to mediate olfactory transduction.

Localization of the sea urchin Spec3 protein to cilia and Golgi complexes of embryonic ectoderm cells.

Expression of the Spec3 gene of Strongylocentrotus purpuratus is associated with ectodermal ciliogenesis. An antiserum was raised against the amino terminus of the deduced Spec3 amino acid sequence and used for immunofluorescent staining. Cilia and an apical structure at the base of the stained cilium of each ectodermal cell stained intensely in gastrula and later stage embryos. Microtubule-depolymerizing agents dispersed the concentrated spot of apical staining, suggesting a localization of Spec3 antigen to the Golgi complex. Immunogold electron microscopy confirmed the localization of Spec3 antigen on cilia and in the Golgi complex. Spec3 antigen showed a diffuse punctate staining pattern in the ectodermal cytoplasm of hatching blastula when Spec3 transcripts are most prevalent, suggesting that after synthesis, Spec3 is sequestered in the Golgi complex before appearing on cilia. Whereas the predicted Mr of the Spec3 protein is 21,600, immunoblotting with S. purpuratus proteins indicated that a Spec3 antigen was concentrated in cilia and migrated as an SDS-resistant aggregate of Mr approximately 350,000. Spec3 is also concentrated in cilia of Lytechinus pictus but the protein migrated with an Mr approximately 23,000 in this species. The S. purpuratus Spec3 antigen remains associated with the ciliary axoneme after extraction of membrane proteins.

Ciliary orientation in the "immotile cilia" syndrome.

Ciliary orientation was studied in 43 patients with the "immotile cilia" syndrome. Twenty-four of these patients had total situs inversus. One mucosal specimen was taken from uterine cervical epithelium, 2 were from bronchial mucosa and 40 from nasal mucosa. The orientation of the cilia was measured from micrographs using a semiautomatic image analyzer (IBAS I). The results from patients were compared with those of 10 control subjects. The mean standard deviation and its standard deviation of the angles of ciliary orientation was 39.7 degrees +/- 9.2 degrees in 43 patients and 27.4 degrees +/- 4.3 degrees in the control group. The difference between the groups is highly significant statistically (P less than or equal to 0.001). However, there were no statistically significant differences in the standard deviations of ciliary orientation between the fields sectioned near the cell membrane or near the ciliary tip. We were also unable to find any significant differences in the standard deviations of the ciliary angles in the specimens taken from brush biopsies and excisional biopsies. There were also no statistically significant differences between the standard deviations of the ciliary angles for the groups with or without situs inversus. If 35 degrees is considered to be the limit value for the mean standard deviation between normal and pathological specimens in our total material, this would give a specificity of 0.90 and a sensitivity of 0.72.

Analysis of mammalian dynein using antibodies against A polypeptides of sea urchin sperm flagellar dynein.

Two different affinity-purified polyclonal antibodies were prepared against A polypeptides of dynein 1 extracted from sea urchin sperm. These antibodies, named AD1 and AD2, reacted exclusively with the alpha and beta heavy chains of dynein 1. Using these antibodies, we analyzed their cross-reactivity with dynein of mammalian cells. Immunohistochemically, both AD1 and AD2 stained dynein-related structures such as cilia of rabbit tracheal epithelia and flagella of rat spermatozoa. Immunoblots of the proteins extracted from mammalian cilia and flagella revealed the presence of A polypeptide-like proteins which cross-reacted with AD1 and AD2. Immunoblot analysis showed that the cross-reactive proteins were localized to the 370-kDa band of rabbit cilia and the 390- and 350-kDa bands of rat sperms. The reaction patterns showed that there were some differences between the two antibodies. On ciliary protein immunoblots, AD1 recognized about half of the broad band region which reacted with AD2, and AD1 also recognized only the 350-kDa band of the flagella extract, suggesting that the antibody reveals only a beta-like polypeptide. Immunoprecipitation studies using the ciliary proteins and AD2 confirmed that the immunoreactive protein had ATPase activity. Given these results, we have characterized mammalian dyneins previously reported by other laboratories.

Immunological detection of spectrin during differentiation and in mature ciliated cells from quail oviduct.

A protein that was immunologically related to the erythrocyte and brain alpha-240-subunit and to the brain beta-235-subunit of spectrin was characterized by immunoblotting and was detected by immunofluorescence in the apical part of ciliated cells from quail oviduct. After immunogold-labeling electron immunocytochemistry, spectrin was detected mainly in a fibrillar meshwork located between the proximal parts of the basal bodies. It was also observed to be in contact with the basal foot of basal bodies, but was not found to be associated with the apical plasma membrane. Cilia and microvilli were unlabeled. In contrast, spectrin was detected in close contact with the lateral plasma membrane of mature ciliated cells as well as in stem epithelial cells in unstimulated oviduct. During ciliogenesis induced by estrogen, spectrin gradually appeared at the apex of the cells as the apical cytoskeleton differentiated.

Immunohistochemical localization of fibronectin in the chinchilla cochlea.

The purpose of this study was to better understand the molecular composition of the cochlea. Fibronectin (FN), a well characterized adhesive glycoprotein, was localized by immunofluorescence microscopy in fresh and fixed cochlear tissues, and in fixed kidney tissue, using a polyclonal, affinity-purified, rabbit, anti-fibronectin antibody and a secondary antibody coupled to FITC. The FN antibody was free from cross-reactivity with other known basement membrane and cell matrix molecules. FN reactivity in the cochlea was most intense in the basilar membrane, latero-basal borders of Boettcher's cells, otic capsule, endothelial basement membranes (particularly those of the stria vascularis), and as a diffuse, fan-shaped network radiating into the spiral ligament. Little FN labelling was present in the epithelial basement membranes. Negative control tissue showed no immunoreactivity; whereas, positive kidney control tissue showed appropriate FN immunoreactivity in the mesangium of the glomerulus. The most significant finding of this study was that FN is a major component of the basilar membrane and its distribution appears to correspond to the amorphous ground substance. FN was not localized in the organ of Corti or at the tips of the hair-cell stereocilia.

The inositol lipids of Paramecium tetraurelia and preliminary characterizations of phosphoinositide kinase activity in the ciliary membrane.

Inositol glycerolipids make up less than 10% of total phospholipids of Paramecium tetraurelia cells. Unlike inositol lipids found in mammalian and other cell types, these lipids from Paramecium lack arachidonic acid. It was demonstrated that kinase and possibly phosphatase enzymes that interconvert phosphatidylinositol (PI), phosphatidylinositol phosphate (PI-P) and phosphatidylinositol-bis-phosphate (PI-P2) exist in ciliary membranes of this ciliate. When exogenous soybean PI and [gamma-32P]ATP were provided as substrates, isolated cilia preparations exhibited PI and PI-P kinase activities as demonstrated by the incorporation of radiolabel into PI-P and PI-P2. Kinase activity was activated by millimolar [Mg2+] and inhibited by millimolar [Ca2+]. Significant inhibition of kinase activity in the presence of unlabeled excess ATP suggested that ATP is the preferred phosphate donor for this reaction. Of 4 suborganellar fractions of isolated cilia, the membrane fraction had the greatest kinase activity indicating that the enzyme(s) is membrane-associated.

Endogenous lectin CSL is present on the membrane of cilia of rat brain ependymal cells.

An endogenous brain lectin, with a great affinity for oligomannosidic glycans, called CSL (for 'cerebellar soluble lectin'), was detected on the surface of the cilia of ependymal cells both in cultures and in vivo. The lectin is not synthesized by the ependymal cells themselves. In vivo it is neither found in cerebrospinal fluid nor in cells of the choroid plexus. Probably, lectin CSL is produced by subependymal astrocytic cells. The membranes of ependymal cells seem to possess glycoprotein ligands for the lectin which explain the specific adhesion of CSL on the surface of these cells, particularly on the cilia. The localization of this adhesive molecule on cilia of ependymal cells suggests that it may play a role in trapping foreign cells, micro-organisms or debris.

Release of intact microtubule-capping structures from Tetrahymena cilia.

The distal ends of ciliary microtubules are attached to the membrane by microtubule-capping structures. The capping structures are located at the sites of tubulin addition and loss in vivo and may be part of the regulatory system that directs ciliary and flagellar microtubule assembly. This study describes conditions for the release and stabilization of microtubule capping structures as a first step in their purification. Two types of capping structures, the distal filaments and the central microtubule caps, are selectively and independently released from the axoneme by CaCl2 and MgCl2 but not by MgSO4, ZnCl2, NaCl, KCl, or KI. The release of the caps and filaments is specific for Ca+2, Mg+2, and Cl- and is not simply a function of ionic strength. The capping structures are released without major disruption of the axonemal structure. In addition to providing a means to purify and identify the cap and filament components, these results suggest ways in which their binding to the axoneme may be modulated during periods of microtubule growth or shortening. This report also reveals that the distal filaments are composed of two separable components, a small bead inserted into the end of each A-tubule and a "Y"-shaped plug and filament that slips through the bead.

Separation of tubulin subunits by reversed-phase high-performance liquid chromatography.

When properly solubilized with trifluoroacetic acid (TFA), alpha- and beta-tubulin subunits from a variety of sources may be resolved at high yield by reversed-phase high-performance liquid chromatography (HPLC), using a Waters muBondapak C18 column and simple linear aqueous acetonitrile gradients containing TFA. The tubulin subunits are typically the most non-polar proteins present, with the beta-tubulin subunit eluting before the alpha. Column temperature above ambient improve both the resolution and the yield; less polar solvent systems do not. Tubulins not freely soluble in aqueous TFA may be solubilized in 6 M guanidine-hydrochloric acid with no change in retention time. Other columns with shorter carbon chain lengths and larger pore size produce a single, unresolved tubulin peak. Reversed-phase HPLC analysis provides an independent comparative evaluation of organelle-specific tubulins, with characteristic retention time differences observed between homologous ciliary and flagellar outer doublet tubulin subunits and also between them and their cytoplasmic counterparts.

Target molecules of calmodulin on microtubules of Tetrahymena cilia.

In the course of an attempt to isolate the calmodulin-binding proteins (CaMBPs) from cilia of Tetrahymena, it was found that some CaMBPs tend to interact with axonemal microtubules. The present study demonstrates this interaction by cosedimentation experiments using in vitro polymerized Tetrahymena axonemal microtubules and Tetrahymena CaMBPs purified from axonemes by calmodulin affinity column chromatography. Analysis by the [125I]calmodulin overlay method showed that at least three CaMBPs (Mr69, 45, and 37 kDa) cosediment with microtubules. Furthermore, without any addition of exogenous CaMBPs, microtubules purified after three cycles of temperature-dependent polymerization and depolymerization included the above CaMBPs and additional CaMBPs (Mr30, 26, and 22 kDa) which could not cosediment with microtubules. From the results, we have classified these microtubule-associated CaMBPs into two groups: (i) CaMBPs which interact with microtubules only during polymerization (30, 26, and 22 kDa), and (ii) CaMBPs which interact not only with microtubules during polymerization, but also with polymerized microtubules (69, 45, and 37 kDa). These results suggest that the microtubule-associated CaMBPs, especially those of the latter group, are located on the surface of ciliary microtubules, and may become the target molecules of calmodulin at Ca2+-triggered ciliary reversal.

Immunogold localization of actin in developing photoreceptor cilia of normal and rds mutant mice.

Recent immunocytochemical studies have localized actin to the distal cilium of mature vertebrate photoreceptors. Since this is the site where the ciliary plasma membrane evaginates to form new outer segment discs, the results suggest that an actin-mediated contractile mechanism or cytoskeletal network might regulate some aspect of outer segment disc morphogenesis. In the present study, immunoelectron microscopy was used to localize actin to the developing cilia of normal and rds mutant mouse photoreceptors. In normal mice, actin could not be localized to newly projecting cilia, but an actin-rich domain was demonstrated within the distal, bulbous ending of elongated cilia just prior to outer segment development. These results suggest that actin is not important for ciliary growth, but that it may be necessary for the subsequent differentiation of an outer segment. In the rds mutant mouse, there is an absence of outer segment formation, although cilia appear to develop normally. Rhodamine phalloidin staining of cryostat sections demonstrated a normal F-actin distribution within the rds retina. Utilizing immunogold labeling of developing rds photoreceptors, actin was localized to the distal, bulbous ending of elongated cilia. This result indicates that actin is situated within its normal domain in rds cilia.

Purification and properties of dyneins from Paramecium cilia.

Dynein ATPases were purified from Paramecium cilia by salt extraction followed by sucrose density gradient centrifugation and anion exchange chromatography. The two major dyneins sedimented in sucrose gradients as species of 22 S and 12 S. After purification by anion exchange chromatography, their specific activities were about 0.4 and 0.5 mumol/min per mg, respectively. The dyneins could be distinguished by subunit composition and immunological crossreactivity. Sucrose density gradient centrifugation revealed additional ATPase activity in the region between the 22 S and 12 S dyneins, including a 19 S activity. Mg2+-ATPase activities of the dyneins and the 19 S activity were inhibited by vanadate and Zn2+, and were activated by Triton X-100. Antibodies against the 22 S dynein from Paramecium reacted on immunoblots with most of the polypeptides of 22 S dynein, and showed that the heavy chains of 22 S dynein are not identical to those that sediment at 19 S and 12 S. Several minor ATPase activities were revealed by anion exchange chromatography of fractions from the 22 S, 19 S and 12 S regions of sucrose gradients. These minor activities were stimulated by Mg2+, inhibited by vanadate, and could be distinguished from each other by their elution positions and polypeptide compositions.

Localization of kinesin in cultured cells.

Kinesin was isolated from bovine brain and used to elicit polyclonal antibodies in rabbits. The specificities of the resulting antibodies were evaluated by immunoblotting. Antibodies purified from these sera by their affinity for brain kinesin react with a polypeptide of approximately 120 kD in extracts from bovine brain, PtK1 cells, and mouse neuroblastoma cells. They bind to a pair of polypeptides of approximately 120 kD present in crude kinesin prepared from Xenopus eggs and with a single polypeptide of approximately 115 kD in extracts from Drosophila embryos. Antibodies raised against kinesin prepared from fruit fly embryos (by W. M. Saxton, Indiana University, Bloomington, IN) and from neural tissues of the squid (by M. P. Sheetz, Washington University, St. Louis, MO) cross react with the mammalian, the fly, and the frog polypeptides. Kinesin antigen was localized in cultured cells by indirect immunofluorescence. PtK1 cells in interphase showed dim background staining of cytoplasmic membranous components and bright staining of a small, fibrous, juxtanuclear structure. Double staining with antibodies to microtubules showed that the fibrous object was usually located near the centrosome. On the basis of shape, size, and location, we identify the kinesin-positive structure as a primary cilium. PtK1 cells in mitosis are stained at their poles during all stages of division. The structure stained is approximately spherical, but wisps of faint fluorescence also extend into the body of the spindle. Antibodies to squid or fruit fly kinesin produce identical patterns in PtK1 cells. Controls with preimmune and preabsorbed sera show that the centrosome staining is not due simply to the common tendency of rabbit antisera to stain this structure. Similar centrosome and spindle pole staining was visible when antibodies to bovine brain or squid kinesin were applied to the A6 cell line (kidney epithelial cells from Xenopus laevis). Some possible functions of kinesin localized at the spindle poles are discussed.

Activation by odorants of a multistate cation channel from olfactory cilia.

Single-channel records were obtained after fusion of ciliary membranes from the olfactory epithelium of Rana catesbeiana to planar lipid bilayers, and odorant-activated cation-selective channels were identified. In addition, a 190-pS potassium-selective channel and a 40-pS cation-selective channel were found in a 0.2 M salt-containing buffer. Odorant-sensitive channels were directly and reversibly activated by nanomolar concentrations of the bell pepper odorant 3-isobutyl-2-methoxypyrazine and the citrus odorant 3,7,-dimethyl-2,6-octadienenitrile. These channels display burst kinetics, multiple conductance levels between 35 and 420 pS, and open times in the millisecond range. With increasing concentrations of odorant, the probability of populating the higher conductance levels increases. These results show that direct activation of channels by odorants may mediate excitation of the olfactory receptor cell.

A cation channel for K+ and Ca2+ from Tetrahymena cilia in planar lipid bilayers.

The single-channel properties for monovalent and divalent cations of a voltage-independent cation channel from Tetrahymena cilia were studied in planar lipid bilayers. The single-channel conductance reached a maximum value as the K+ concentration was increased in symmetrical solutions of K+. The concentration dependence of the conductance was approximated to a simple saturation curve (a single-ion channel model) with an apparent Michaelis constant of 16.3 mM and a maximum conductance of 354 pS. Divalent cations (Ca2+, Ba2+, Sr2+, and Mg2+) also permeated this channel. The sequence of permeability determined by zero current potentials at high ionic concentrations was Ba2+ greater than or equal to K+ greater than or equal to Sr2+ greater than Mg2+ greater than Ca2+. Single-channel conductances for Ca2+ were nearly constant (13.9 pS-20.5 pS) in the concentrations between 0.5 mM and 50 mM Ca-gluconate. In the experiments with mixed solutions of K+ and Ca2+, a maximum conductance of Ca2+ (gamma Camax) and an apparent Michaelis constant of Ca2+ (K Cam) were obtained by assuming a simple competitive relation between the cations. Gamma Camax and K Cam were 14.0 pS and 0.160 mM, respectively. Single-channel conductances in mixed solutions were well-fitted to this competitive model supporting that this cation channel behaves as a single-ion channel. This channel had relatively high-affinity Ca2+-binding sites.

Cytoskeletal architecture and immunocytochemical localization of fodrin in the terminal web of the ciliated epithelial cell.

In order to understand the cytoskeletal architecture at the terminal web of the ciliated cell, we examined chicken tracheal epithelium by quick-freeze deep-etch (QFDE) electron microscopy combined with immunocytochemistry of fodrin. At the terminal web, the cilia ended into the basal bodies and then to the rootlets. The rootlets were composed of several filaments and globular structures attached regularly to them. Decoration with myosin subfragment 1 (S1) revealed that some actin filaments ran parallel to the apical plasma membrane between the basal bodies, and other population traveled perpendicularly or obliquely, i.e., along the rootlets. Some actin filaments were connected to the surface of the basal bodies and the basal feet. Among the basal bodies and the rootlets there existed three kinds of fine crossbridges, which were not decorated with S1. In the deeper part of the terminal web, intermediate filaments were observed between the rootlets and were sometimes crosslinked with the rootlets. Immunocytochemistry combined with the QFDE method revealed that fodrin was a component of fine crossbridges associated with the basal bodies. We concluded that an extensive crosslinker system among the basal bodies and the rootlets along with networks of actin and intermediate filaments formed a structural basis for the effective beating of cilia.

Structure and mass analysis of 14S dynein obtained from Tetrahymena cilia.

Scanning transmission electron microscopic analysis revealed that the 14S fraction of Tetrahymena dynein was of a mixture of two types of particles in approximately equal proportions. The 14S dynein molecules were roughly ellipsoid in shape with approximate axes of 9.5 and 14.5 nm. About half of the particles had tails 20-24-nm long. By the integration of electron scattering intensities, particles with tails had an average mass of 510 kD with a SD of 90 kD. The globular heads of both types of particles had an average mass of 330 kD with a SD of 60 kD. The mass of the tail structure was about 180 kD. By SDS-PAGE, the 14S dynein consisted of two high molecular mass polypeptides above 300 kD that could be distinguished by immunoblot analysis.

Structure and mass of mammalian respiratory ciliary outer arm 19S dynein.

Mammalian respiratory ciliary outer arm dyneins isolated as the major ATPase peak migrating at 19S on sucrose density gradients were examined by transmission electron microscopy of negatively stained samples and scanning transmission electron microscopy of unstained samples. The predominant discrete particle structure observed was composed of two globular heads apparently connected by amorphous or indistinct material. The heads were either circular or slightly elliptical of mean 13 +/- 1 X 10 +/- 2 nm dimensions. The mass of this structure averaged 1.22 +/- 0.34 million daltons with the individual globular heads averaging 310 +/- 77 kilodaltons (kD). Negative staining revealed that one or both of the globular heads often contained a central accumulation of stain measuring 2.5 +/- 1 nm across. A second type of structure, appearing with lesser frequency in the 19S fraction than in the unfractionated dynein preparation loaded onto the sucrose gradient, was a single globular head of 13 +/- 1 X 10 +/- 2 nm often with 2 +/- 1 nm centrally accumulated stain and with or without an appendage. This one-headed particle thus resembled one-half of the two-headed particle. Mass measurements were lower, however, for isolated, single globular heads, averaging 220 +/- 111 kD. A third type of particle observed was a ring-like structure with 4 +/- 1 nm centrally accumulated stain and without appendages. The ring structure was slightly larger in diameter, 14 +/- 1 nm, and had a greater peripheral accumulation of negative stain than either of the one- or two-headed particles, suggesting that it was not derived therefrom.(ABSTRACT TRUNCATED AT 250 WORDS)