Ciliary microtubule capping structures contain a mammalian kinetochore antigen

Structures that cap the plus ends of microtubules may be involved in the regulation of their assembly and disassembly. Growing and disassembling microtubules in the mitotic apparatus are capped by kinetochores and ciliary and flagellar microtubules are capped by the central microtubule cap and distal filaments. To compare the ciliary caps with kinetochores, isolated Tetrahymena cilia were stained with CREST (Calcinosis/phenomenon esophageal dysmotility, sclerodactyly, telangiectasia) antisera known to stain kinetochores. Immunofluorescence microscopy revealed that a CREST antiserum stained the distal tips of cilia that contained capping structures but did not stain axonemes that lacked capping structures. Both Coomassie blue-stained gels and Western blots probed with CREST antiserum revealed that a 97-kD antigen copurifies with the capping structures. Affinity-purified antibodies to the 97-kD ciliary protein stained the tips of cap-containing Tetrahymena cilia and the kinetochores in HeLa, Chinese hamster ovary, and Indian muntjak cells. These results suggest that at least one polypeptide found in the kinetochore is present in ciliary microtubule capping structures and that there may be a structural and/or functional homology between these structures that cap the plus ends of microtubules.     

Transmembrane currents in frog olfactory cilia

Summary We have measured transmembrane currents in intact single cilia from frog olfactory receptor neurons. A single cilium on a neuron was sucked into a patch pipette, and a high-resistance seal was formed near the base of the cilium. Action potentials could be induced by applying suction or a voltage ramp to the ciliary membrane. A transient current was seen in some cells on stimulation with odorants. After excision from the cell, most of the cilia showed increased conductance in a bath containing cAMP, indicating that the cytoplasmic face of the ciliary membrane was accessible to the bath. The estimated resistance of a single cilium was surprisingly low.     

Subunits of the chaperonin CCT are associated with Tetrahymena microtubule structures and are involved in cilia biogenesis

The cytosolic chaperonin CCT is a heterooligomeric complex of about 900 kDa that mediates the folding of cytoskeletal proteins. We observed by indirect immunofluorescence that the Tetrahymena TpCCTalpha, TpCCTdelta, TpCCTepsilon, and TpCCTeta-subunits colocalize with tubulin in cilia, basal bodies, oral apparatus, and contractile vacuole pores. TpCCT-subunits localization was affected during reciliation. These findings combined with atomic force microscopy measurements in reciliating cells indicate that these proteins play a role during cilia biogenesis related to microtubule nucleation, tubulin transport, and/or axoneme assembly. The TpCCT-subunits were also found to be associated with cortex and cytoplasmic microtubules suggesting that they can act as microtubule-associated proteins. The TpCCTdelta being the only subunit found associated with the macronuclear envelope indicates that it has functions outside of the 900 kDa complex. Tetrahymena cytoplasm contains granular/globular-structures of TpCCT-subunits in close association with microtubule arrays. Studies of reciliation and with cycloheximide suggest that these structures may be sites of translation and folding. Combined biochemical techniques revealed that reciliation affects the oligomeric state of TpCCT-subunits being tubulin preferentially associated with smaller CCT oligomeric species in early stages of reciliation. Collectively, these findings indicate that the oligomeric state of CCT-subunits reflects the translation capacity of the cell and microtubules integrity.     

Properties of microtubule sliding disintegration in isolated tetrahymena cilia

Properties of the sliding disintegration response of demembranated tetrahymena cilia have been studied by measuring the spectrophotomeric response or turbidity of cilia suspensions at a wavelength of 350 nm relative to changes in the dynein substrate (MgATP(2-)) concentration. The maximum decrease in turbidity occurs in 20 muM ATP, and 90 percent of the decrease occurs in approximately 5.9 s. At lower ATP concentrations (1-20 muM), both the velocity and magnitude of the turbidity decreases are proportional to ATP concentration. The velocity data for 20 muM ATP permit construction of a reaction velocity curve suggesting that changes in turbidity are directly proportional to the extent and velocity of disintegration. At ATP concentrations more than 20 muM (50muM to 5mM), both velocity and magnitude of the turbidimetric response are reduced by approximately 50 percent. This apparent inhibition results in a biphasic response curve that may be related to activation of residual shear resistance or regulatory components at the higher ATP concentrations. The inhibitory effects of elevated ATP can be eliminated by mild trypsin proteolysis, whereupon the reaction goes to completion at any ATP concentration. The turbidimetric responses of the axoneme-substrate suspensions are consistent with the extent and type of axoneme disintegration revealed by electron microscope examination of the various suspensions, suggesting that the turbidimetric assay may prove to be a reliable means for assessing the state of axoneme integrity.     

Cyclic AMP diffusion coefficient in frog olfactory cilia

Cyclic AMP (cAMP) is one of the intracellular messengers that mediate odorant signal transduction in vertebrate olfactory cilia. Therefore, the diffusion coefficient of cAMP in olfactory cilia is an important factor in the transduction of the odorous signal. We have employed the excised cilium preparation from the grass frog (Rana pipiens) to measure the cAMP diffusion coefficient. In this preparation an olfactory cilium is drawn into a patch pipette and a gigaseal is formed at the base of the cilium. Subsequently the cilium is excised, allowing bath cAMP to diffuse into the cilium and activate the cyclic nucleotide-gated channels on the plasma membrane. In order to estimate the cAMP diffusion coefficient, we analyzed the kinetics of the currents elicited by step changes in the bath cAMP concentration in the absence of cAMP hydrolysis. Under such conditions, the kinetics of the cAMP-activated currents has a simple dependence on the diffusion coefficient. From the analysis we have obtained a cAMP diffusion coefficient of 2.7 +/- 0.2. 10(-6) cm2 s-1 for frog olfactory cilia. This value is similar to the expected value in aqueous solution, suggesting that there are no significant diffusional barriers inside olfactory cilia. At cAMP concentrations higher than 5 microM, diffusion slowed considerably, suggesting the presence of buffering by immobile cAMP binding sites. A plausible physiological function of such buffering sites would be to prolong the response of the cell to strong stimuli.     

Calcium-dependent phosphorylation of a protein in the frog olfactory cilia

In the cilia of vertebrate olfactory sensory neurons, cytoplasmic Ca(2+) concentration increases in response to odorant stimulation, and this increase has been implicated to have important roles in the regulation of olfactory responses. Since protein phosphorylation is often a regulatory mechanism of biological reactions, we explored the effect of Ca(2+) on phosphorylation reactions in the frog olfactory cilia. First, we found that a 45-kDa phosphoprotein (p45) is predominantly phosphorylated in vitro in the isolated cilia in a Ca(2+)-dependent manner. However, later studies showed that the phosphorylation level of p45 is controlled by a dynamic equilibrium between phosphorylation and dephosphorylation. Although both activities are enhanced at high Ca(2+) concentrations (K(1/2) = approximately 2 microM in both reactions), the enhancement of dephosphorylation is relatively greater than that of phosphorylation. As a result, the steady phosphorylation level of p45 is lower at high than at low Ca(2+) concentration. The phosphorylation/dephosphorylation equilibrium was founed to involve protein kinases sensitive to zinc and heparin, and an unknown phosphatase(s). The present result suggests the presence of a novel Ca(2+)-signaling pathway that involves phosphorylation of p45 in the olfactory cilia.     

Two molecular motility systems of the frog olfactory cilia

Intravital video microscopy was used to study the motility of frog (Rana temporaria) olfactory cilia exposed to various odorants—pentanol, camphor, cineole, and vanillin (first group); ammonia and hydrogen sulfide (second group)—and to the cell respiration inhibitors rotenone and malonate. It was demonstrated that the olfactory cilia had both the dynein-tubulin and actin-myosin molecular motility systems, the former providing unordered and the latter, ordered movements. The motility became ordered in response to exposure to odorants. The tested odorants belonging to different groups had different effects on the mitochondrial respiratory chain activity and the motility of olfactory cilia.     

Comprehensive interaction of dicalcin with annexins in frog olfactory and respiratory cilia

Dicalcin (renamed from p26olf) is a dimer form of S100 proteins found in frog olfactory epithelium. S100 proteins form a group of EF-hand Ca(2+)-binding proteins, and are known to interact with many kinds of target protein to modify their activities. To determine the role of dicalcin in the olfactory epithelium, we identified its binding proteins. Several proteins in frog olfactory epithelium were found to bind to dicalcin in a Ca(2+)-dependent manner. Among them, 38 kDa and 35 kDa proteins were most abundant. Our analysis showed that these were a mixture of annexin A1, annexin A2 and annexin A5. Immunohistochemical analysis showed that dicalcin and all of these three subtypes of annexin colocalize in the olfactory cilia. Dicalcin was found to be present in a quantity almost sufficient to bind all of these annexins. Colocalization of dicalcin and the three subtypes of annexin was also observed in the frog respiratory cilia. Dicalcin facilitated Ca(2+)-dependent liposome aggregation caused by annexin A1 or annexin A2, and this facilitation was additive when both annexin A1 and annexin A2 were present. In this facilitation effect, the effective Ca(2+) concentrations were different between annexin A1 and annexin A2, and therefore the dicalcin-annexin system in frog olfactory and respiratory cilia can cover a wide range of Ca(2+) concentrations. These results suggested that this system is associated with abnormal increases in the Ca(2+) concentration in the olfactory and other motile cilia.     

Gated conductances in native and reconstituted membranes from frog olfactory cilia.

Although cAMP is well established as a second messenger for olfactory transduction in vertebrates, the role of inositol 1,4,5-trisphosphate (IP3) in this process remains controversial. We addressed this issue by comparing currents evoked by cAMP and IP3 in native and reconstituted membranes from olfactory cilia. We detected only a cyclic nucleotide-gated conductance in the native membrane but both cyclic nucleotide-gated and IP3-gated conductances in the reconstituted membrane. The magnitudes of the cyclic nucleotide- and IP3-gated conductances were not correlated with each other in reconstituted membranes, suggesting that cyclic nucleotide- and IP3-gated channels originate in different cellular compartments.     

NEM tubulin inhibits microtubule minus end assembly by a reversible capping mechanism

Although microtubule (MT) dynamic instability is thought to depend on the guanine nucleotide (GTP vs GDP) bound to the beta-tubulin of the terminal subunit(s), the MT minus end exhibits dynamic instability even though the terminal beta-tubulin is always crowned by GTP-alpha-tubulin. As an approach toward understanding how dynamic instability occurs at the minus end, we investigated the effects of N-ethylmaleimide-modified tubulin (NTb) on elongation and rapid shortening of individual MTs. NTb preferentially inhibits minus end assembly when combined with unmodified tubulin (PCTb), but the mechanism of inhibition is unknown. Here, video-enhanced differential interference contrast microscopy was used to observe the effects of NTb on MTs assembled from PCTb onto axoneme fragments. MTs were exposed to mixtures of PCTb (25 microM) and NTb (labeled on approximately 1 Cys per monomer) in which the NTb/PCTb ratio varied from 0.025 to 1. The NTb/PCTb mixture had a slight inhibitory effect on the plus end elongation rate, but significantly inhibited or completely arrested minus end elongation. For the majority of mixtures that were assayed (0.1-1 NTb/PCTb ratio), minus end MT length remained constant until the NTb/PCTb mixture was replaced. Replacement with PCTb allowed elongation to proceed, whereas replacement with buffer or NTb caused minus ends to shorten. Taken together, the results indicate that NTb associates with both plus and minus ends and that NTb acts to reversibly cap minus ends only when PCTb is also present. Low-resolution mapping of labeled Cys residues, along with previous experiments with other Cys-reactive compounds, suggests that modification of beta-tubulin Cys(239) may be associated with the capping action of NTb.     

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.     

Non-ependymal cilia in the habenulae and the interpeduncular nucleus of the frog tadpole

Summary Cilia of the 9+2 pattern are found electron microscopically in nonependymal cells of the habenulae and the interpeduncular nucleus of the tadpole of Rana esculenta at an early stage of development (8 mm length, head to tip of tail). A comparison is made between these and the ependymal and sensory cilia in the same specimens. The cilia project into the neuropil emerging from a perikaryon rich in free ribosomes and displaying a prominent Golgi apparatus. These perikarya contain dense core vesicles. Synapses with vesicles of the clear spherical type have been observed along the ciliary shaft. On a purely morphologic basis the authors hypothesize that these cilia, at least in this early ontogenetic stage, may extend considerably the conducting surface of the cell and represent a sensory structure which could be stimulated by terminal processes belonging to distantly located cells. In addition, they could also be involved in the trophic exchange of material with the adjacent structures.     

Plastid microtubule-like structures in wheat are insensitive to microtubule inhibitors

The effects of microtubule inhibitors on the spectral properties of leaves of wheat ( Triticum aestivum L. cv. Walde) and on the presence of plastid microtubule–like structures (MTLS) during etioplast to chloroplast transformation were examined. Amiprophos-methyl (APM, 0.1 m M ), fed to leaf sections of 7-day-old dark-grown wheat, reduced the ration of phototransformable to non-phototransformable proto-chlorophyllide (PChlide), decreased the rate of the Shibata shift, and inhibited chlorophyll accumulation and grana stacking. The spectral properties of isolated etioplasts were not affected by APM. Colchicine (10 m M ), fed to leaf sections, inhibited greening but had no effect on the PChlide ratio or the Shibata shift. MTLS were still visible on electron micrographs after treatment with APM or colchicine at frequencies similar to controls. A third inhibitor, vinblastine, had no effect on the spectral properties of non-irradiated or irradiated etiolated leaves except at concentrations that produced visible tissue damage before the irradiation. The effects of APM and colchicine may reflect inhibitions of respiration and protein synthesis, respectively. It is concluded that MTLS are insensitive to microtubule inhibitors and thus are probably not composed of tubulin.     

Tracheal motile cilia in mice require CAMSAP3 for the formation of central microtubule pair and coordinated beating

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body (BB), which are linked by a “transition zone” (TZ). The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a protein that can stabilize the minus-end of a microtubule, concentrates at multiple sites of the cilium–BB complex, including the upper region of the TZ or the axonemal basal plate (BP) where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the BP, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme and thereby supports the coordinated motion of multicilia in airway epithelial cells.     

Mucus-depleted frog palate as a model for the study of mucociliary clearance

To better understand the frog palate model of mucociliary transport, we measured the transport rate of mucus (MTR) from the leopard frog, Rana pipiens, and from the bullfrog, R. catesbeiana, recorded the stability of the MTR over a period of hours and days and over the course of 1 yr, and measured the viscoelasticity, percent solid composition, and spinnability (filance) of mucus from both species. Bullfrog mucus was less rigid than leopard frog mucus (log G* at 1 rad/s 2.09 vs. 2.61; P less than 0.01) and had a higher viscosity-to-elasticity ratio (tan delta at 1 rad/s 0.36 vs. 0.26; P less than 0.05). It also had a lower solids content (8.71 vs. 13.72%; P = 0.02), and there was a trend to lower spinnability for bullfrog mucus (filance 26.7 vs. 33.5 mm). These data suggest that bullfrog mucus has viscoelastic properties similar to normal mammalian respiratory mucus and leopard frog mucus has viscoelasticity similar to sputum samples. MTR was significantly slower in the winter than in the summer months (17 vs. 30 mm/min; P less than 0.0001). Although the leopard frog palate could be used for at least 7 consecutive days without exhaustion, bullfrog palates could be used for only 5 days. Palates of either species could generally be tested for 6 h/day without a significant decrease in MTR. These data clarify some of the sources of variability in the use of this system and suggest methods of standardization.