Protein differences in cilia of mechanoreceptor hair and gill cells of the scallop Mizuhopecten yessoensis

• 1.1. In search for mechanosensory molecules the composition of the ciliary proteins of mechanoreceptor hair cells of the abdominal organ and less mechanosensitive gill cells were compared electrophoretically.
• 2.2. The hair cells and gill cilia were very similar in their polypeptide sets but differed by contents of three axonemal polypeptides with molecular weights of 125, 149 and 300 kilodaltons (kDa) and one membrane polypeptide of 159 kDa.
• 3.3. The membrane polypeptide with a molecular weight of 159 kDa represented approximately 3% of the total ciliary protein of hair cells. There was only a trace of this polypeptide in gill cilia and their membrane fraction.
• 4.4. A peculiarity of ciliary membranes of the hair cells was a high content of the 159 kDa-polypeptide, which constituted more than 20% of the total protein of membrane fraction.

     

Studies on calmodulin isolated from Tetrahymena cilia and its localization within the cilium

Tetrahymena calmodulins from cilia, cell bodies and whole cells were isolated separately and compared. These calmodulins showed just the same properties: they co-migrated in SDS-polyacrylamide gel electrophoresis, had a Ca²⁺-dependent electrophoretic mobility change in alkali gel, held the same antigenic determinants in common, and activated brain cyclic nucleotide phosphodiesterase Ca²⁺-dependently with identical activation curves. Distributions of calmodulin and calmodulin-counterpart in Tetrahymena cilium were investigated by using alkali gel electrophoresis in the presence of Ca²⁺ or EGTA, and by immunoelectron microscopy. Calmodulin was detected in the membrane plus matrix fraction and outer-doublet microtubule fraction, and its Ca²⁺-dependent counterpart existed exclusively in the latter fraction. However, neither calmodulin nor its counterpart was detected in the crude dynein fraction. Immunoelectron microscopy revealed that calmodulin was localized along the longitudinal axis of outer-doublet microtubules at regular intervals of about 90 nm. The calmodulin-binding site in the ciliary axoneme was suggested to be interdoublet links.     

Nitric oxide-dependent cilia regulatory enzyme localization in bovine bronchial epithelial cells.

Airway epithelial-derived nitric oxide (NO), through the activation of nucleotide cyclases and downstream kinases, stimulates ciliary beating, yet the precise locations of these enzymes are unknown. We hypothesized that these NO-activated enzymes are located within, or adjacent to, the ciliary axoneme. Immunohistochemistry of intact ciliated cells revealed that endothelial-type nitric oxide synthase (eNOS), the RII isoform of the cAMP-dependent protein kinase (PKA-RII), the type I isoform of the cGMP-dependent protein kinase (PKG-I), and guanylate cyclase beta (GC-beta) all colocalized with pericentrin to the basal body. In contrast, the PKA-RI isoform and the PKG-II isoform localized to ciliary axonemes. Western blot analysis of isolated demembranated ciliary preparations detected eNOS, GC-beta, and both isoforms of PKA and PKG. An A-kinase-anchoring protein was also detected. Our findings suggest that these enzymes are sequestered close to their points of action into a discrete ciliary metabolon, enabling targeted phosphorylation and efficient upregulation of ciliary beating.     

Laser-induced spreading arrest of Mytilus gill cilia

Using a "slit camera" recording technique, we have examined the effects of local laser irradiation of cilia of the gill epithelium of Mytilus edulis. The laser produces a lesion which interrupts epithelial integrity. In artificial sea water that contains high K+ or is effectively Ca++ free, metachronism of the lateral cilia continues to either side of the lesion with only minor perturbations in frequency synchronization and wave velocity, such as would be expected if metachronal wave coordination is mechanical. However, in normal sea water and other appropriate ionic conditions (i.e., where Ca++ concentration is elevated), in addition to local damage, the laser induces distinct arrest responses of the lateral cilia. Arrest is not mechanically coordinated, since cilia stop in sequence depending on stroke position as well as distance from the lesion. The velocity of arrest under standard conditions is about 3 mm/s, several orders of magnitude faster than spreading velocities associated with diffusion of materials from the injured region. Two responses can be distinguished on the basis of the kinetics of recovery of the arrested regions. These are (a) a nondecremental response that resembles spontaneous ciliary stoppage in the gills, and (b) a decremental response, where arrest nearer the stimulus point is much longer lasting. The slower recovery is often periodic, with a step size approximating lateral cell length. Arrest responses with altered kinetics also occur in laterofrontal cilia. The responses of Mytilus lateral cilia resemble the spreading ciliary arrest seen in Elliptio and arrest induced by electrical and other stimuli, and the decremental response may depend upon electrotonic spread of potential change produced at the stimulus site. If this were coupled to transient changes in Ca++ permeability of the cell membrane, a local rise in Ca++ concentration might inhibit ciliary beat at a sensitive point in the stroke cycle to produce the observed arrest.     

Calcium-dependent phosphatidylinositol phosphorylation in lamellibranch gill lateral cilia

Summary Pure lateral (L) cilia may be separated from the remaining (R) cilia types ofMytilus edulis gill by serotonin activation after hypertonic shock. The two classes of cilia were permeabilized with 0.012% Triton X-100 and incubated with³²P-labeled ATP at low Ca⁺⁺ (10^–7 M), where L cilia beat, or in high Ca⁺⁺ (2–20 M), where L cilia arrest but R cilia are active. The labeled cilia were separated into axoneme and membrane-matrix fractions by detergent extraction, subjected to SDS-PAGE on 5–15% gels, and autoradio-graphed. Neither cilia type undergoes Ca⁺⁺-dependent phosphorylation of specific proteins, suggesting that neither Ca⁺⁺-induced arrest in L cilia nor the Ca⁺⁺ activation of other cilia is phosphorylation-dependent. However, lipid phosphorylation in L cilia is highly Ca⁺⁺-dependent. Identified by thin-layer chromatography, the phospholipid that is phosphorylated in a Ca⁺⁺-dependent manner is phosphatidylinositol 4-phosphate (PIP), yielding the 4,5-bisphosphate (PIP₂). PIP₂ increases at least 3-fold under Ca⁺⁺-arrest conditions.Aequipecten gill lateral cilia, which require higher Ca⁺⁺ levels for arrest, show even more striking changes. In both cases, the effect is maximal at micromolar Ca⁺⁺ levels. Phosphorylation of other lipids is Ca⁺⁺-independent. In the Ca⁺⁺-insensitive or activated R cilia, PIP₂ levels are intermediate, increasing only marginally with increased [Ca⁺⁺]. The formation of PIP₂ in response to Ca⁺⁺, as opposed to its breakdown to form inositol 1,4,5-trisphosphate and diacylglycerol, may be characteristic of a Ca⁺⁺ transport system. Mechanically sensitive, the L cilia arrest as a consequence of an inward flux of Ca⁺⁺ ions, acting directly on the axoneme. After Ca⁺⁺-induced arrest, the formation of PIP₂ may be involved in sequestering Ca⁺⁺ or in augmenting Ca⁺⁺ pump activity, thus reducing Ca⁺⁺ levels so that motility may resume quickly.     

Immunocytochemical localization of tubulin with colloidal gold in cilia of rabbit tracheal epithelial cultures

Ciliated tracheal epithelia cell cultures were investigated immunocytochemically with anti-tubulin and colloidal gold. When rabbit tracheal cultures were fixed in paraformaldehyde, treated with acetone, anti-tubulin and a second antibody coupled to FITC, fluorescence was associated with cytoskeletal and axonemal microtubules. Cilia covering the apical surface of the ciliated tracheal cells fluoresced very brightly thus facilitating identification of this cell type. Electron microscopy of tracheal cultures fixed as above, treated with Triton-X 100 and incubated in anti-tubulin and protein A coupled to colloidal gold resulted in the highly specific localization of tubulin in ciliary axonemes and basal bodies. Omission of primary or secondary antibody resulted in extremely low levels of fluorescence while no colloidal gold particles could be detected in cultures at the electron microscopy level when rabbit anti-tubulin was omitted.     

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.     

Cyclic AMP and calcium in the differential control ofMytilus gill cilia

Summary Lateral (L) cilia ofMytilus gill are activated by serotonin which, in molluscan systems, is known to activate adenylate cyclase. Triton-extracted models of L-cells, arrested at >10^–6 M Ca⁺⁺, are stimulated to beat by the addition of 10^–5 M cAMP while still under Ca⁺⁺ arrest conditions, suggesting that cAMP-activation is not mediated by alterations of Ca⁺⁺ levels. Using isolated, permeabilized cilia, we find, independent of [Ca⁺⁺], that cAMP-dependent protein phosphorylation in L-cilia occurs uniquely and reversibly on three low molecular weight polypeptides of 23,000, 18,000, and 14,000 daltons. Phosphorylation is maximal at cAMP concentrations above 0.5 M. The phosphorylated chains partially coextract at high salt with a 14S dynein fraction and have approximately the same molecular weights as reported for dynein light chains. Such conditions mainly extract the outer dynein arm, about 40% of the Mg⁺⁺-ATPase activity, and a corresponding amount of the cAMP phosphorylated chains. However, the three polypeptides sediment together at 10–11S, clearly separable from the 14S dynein ATPase. Using a gel-overlay technique, we find that calmodulin binds to axonemal polypeptides of L-cilia with molecular weights of 18,000 and 13,000, independent of Ca⁺⁺, while in mixed-population cilia, only a 12,000 dalton chain binds calmodulin, in a Ca⁺⁺ dependent manner. In neither case are calmodulin binding proteins found in the high salt fraction containing the cAMP-dependent phosphorylated chains, indicating that, in spite of some similarity in molecular weight, the cAMP-phosphorylated and calmodulin binding polypeptides are different. Also, double-labeling indicates that only the 18,000 dalton chains co-migrate. These data suggest that serotonin may activate lateral cilia through a cAMP-dependent phosphorylation of a dynein-associated regulatory protein complex, while Ca⁺⁺ may inhibit ciliary movement, independently, by binding to calmodulin associated with a different class of regulatory protein.     

Effect of vanadate on gill cilia: Switching mechanism in ciliary beat

Lateral (L) cilia of freshwater mussel (Margaritana margaritifera and Elliptio complanatus) gills can be arrested in one of two unique positions. When treated with 12.5 mM CaCl₂ and 10^?5 M A23187 they arrest in a “hands up” position, ie, pointing frontally. When treated with approximately 10 mM vanadate (V) they arrest in a “hands down” position, ie, pointing abfrontally. L-cilia treated with 12.5 mM CaCl₂ and 1 mM NaN₃ also arrest in a “hands down” position; substitution of 20 mM KC1 and 1 mM NaN₃ causes cilia to move rapidly and simultaneously to a “hands up” position. The observations suggest that there are two switching mechanisms for activation of active sliding in ciliary beat one at the end of the recovery stroke and the other at the end of the effective stroke; the first is inhibited by calcium and the second by vanadate or azide. This is consistent with a model of ciliary beating where microtubule doublet numbers 1, 2, 3, and 4 are active during the effective stroke while microtubule doublets numbers 6, 7, 8, and 9 are passive, and the converse occurs during the recovery stroke.     

Immunohistochemical localization of calmodulin in mouse brain

Calmodulin is a well-known calcium-binding protein which is ubiquitous in the plant and animal kingdoms and regulates many cellular processes. In this paper, the distribution of calmodulin in mouse brain was studied immunohistochemically using specific anti-calmodulin IgG which was raised in the rabbit by immunization with native calmodulin. Immunoreactive staining was observed in the cells in almost all areas of the brain, but its intensity varied. Some areas were stained heavily even in the presence of high concentration of NaCl. These differed from those stained immunohistochemically with antibody against other calcium-binding proteins, parvalbumin or vitamin D-dependent calcium-binding protein.     

Tetrahymena calmodulin. Characterization of an anti-tetrahymena calmodulin and the immunofluorescent localization in Tetrahymena

A rabbit antiserum specific for Tetrahymena calmodulin was prepared and characterized: In Ouchterlony's immunodiffusion test, the antiserum gave rise to a single precipitin line only with calmodulin in the reaction with crude Tetrahymena extract and the antiserum cross-reacted with a calmodulin fraction from Paramecium, but not with several calmodulin fractions, from higher organisms. Calmodulins from the ciliates appear to share some antigenic determinants which are absent in calmodulins from higher organisms. The intracellular localization of calmodulin was investigated by indirect immunofluorescent method using anti-Tetrahymena calmodulin antibody purified on an antigen-Sepharose affinity column. Immunofluorescence was localized in the oral apparatus, cilia, basal bodies, the anterior end of the cell, and the contractile vacuole pores. The localization suggested involvement of calmodulin in food vacuole formation (nutrient uptake), excretion of contractile vacuole contents (regulation of osmotic pressure), and in ciliary movement (reversal). The suggestion was supported by the observation that trifluoperazine markedly suppressed food vacuole formation and excretion of contractile vacuole contents and affected the ciliary motion.     

Heterogeneous localization of epitopes along axonemes of mammalian cilia

The specificity of four monoclonal antibodies, raised against mammalian ciliary axonemes, was determined by both immunofluorescence and immunoblot experiments. Three antibodies reacted with epitopes which are differentially located along axonemal length. Among these, antibody 3.12 recognized an epitope common to different dynein heavy chains, reacted only with tracheal cilia and specifically stained the proximal portion of the ciliary axoneme.     

Immunoelectron microscopic localization of calmodulin in corn root cells

Methods for the localization of plant calmodulin by immuno-gold and immuno-peroxidase electron microscopy have been developed. In both corn root-cap cells and meristematic cells, calmodulin was found to be localized in the nucleus, cytoplasm, mitochondria as well as in the cell wall, In the meristematic cells, calmodulin was distinctly localized on the plasma membrane, cytoplasmic face of rough endoplasmic rcticulum and polyribosomes. Characteristically, calmodulin was present in the amyloplasts of root-cap cells. The widespread distribution of calmodulin may reflect its plciotropic functions in plant cellular activities.     

Homologues of radial spoke head proteins interact with Ca2+/calmodulin in Tetrahymena cilia

Calmodulin (CaM) is an axonemal component. To examine the pathway of Ca(2+)/CaM signaling in cilia, using Ca(2+)/CaM-affinity column, we identified seven Ca(2+)/CaM-associated proteins from a crude dynein fraction and isolated 62 kDa (p62) and 66 kDa (p66) Ca(2+)/CaM-associated proteins in Tetrahymena cilia. The amino acid sequences deduced from the p62 and p66 cDNA sequences suggested that these proteins were similar to Chlamydomonas radial spoke proteins 4 and 6 (RSP4 and RSP6), components of the radial spoke head, and sea urchin sperm p63, which is a homologue of RSP4/6, and isolated as a key component that affect flagellar bending patterns. Although p62 and p66 do not have a conventional CaM-binding site, those have consecutive sequences which showed high normalized scores (>or= 5) from a CaM target database. These consecutive sequences were also found in RSP4, RSP6, and p63. These radial spoke heads proteins have a high similarity region composed of 15 amino acids between the five proteins. Immunoelectron microscopy using anti-CaM antibody showed that CaM was localized along the outer edge of the curved central pair microtubules in axoneme. Therefore, it is possible that the interaction between Ca(2+)/CaM and radial spoke head control axonemal curvature in the ciliary and flagellar waveform.     

Immunocytochemical localization of piscidin in mast cells of infected seabass gill

Annual losses of ～5–10% of the juvenile stock of European seabass, Dicentrarchus labrax (L.) in the northern coast of the Adriatic Sea has been attributed to heavy infections of the gill monogenean Diplectanum aequans. Immunocytochemical, light and ultrastructural studies were carried out on seabass naturally parasitized with this monogenean. The site of the worm's attachment was marked by the common presence of haemorrhages and white mucoid exudate. In histological sections, infected gills showed hyperplasia, as well as proliferation of mucous cells and rodlet cells. Disruption and fusion of the secondary lamellae were common in all infected seabass, with several specimens also showing marked inflammation and erosion of the primary and secondary lamellar epithelium. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of mast cells that were positive. Mast cells were both within and outside the blood vessels of the primary and secondary lamellae, and often made intimate contact with vascular endothelial cells. Mast cells were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Our data provide evidence showing the presence of piscidin 3 in the cytoplasmic granules of an important group of fish inflammatory cells, the mast cells resident in seabass gill tissue. There was no significant difference in the number of HAGR-positive mast cells between infected and uninfected fish (ANOVA, p > 0.05). However, mast cells in parasitized gills usually showed much stronger immunostaining intensity compared to those in unparasitized gills. These data are the first to document a response of piscidins or any other antimicrobial peptide of fish to parasite infection and suggest that mast cells may play a role in certain inflammatory responses without a detectable increase in their numbers.     

Presence and indirect immunofluorescent localization of calmodulin in Paramecium tetraurelia

In this paper we demonstrate the presence and localization of calmodulin, a calcium-dependent regulatory protein, in the ciliated protozoan Paramecium tetraurelia. Calmodulin is demonstrated by several criteria: (a) the ability of whole cell Paramecium extracts to stimulate mammalian phosphodiesterase activity, (b) the presence of an acidic, thermostable, 17,000-dalton polypeptide whose mobility shifts in SDS polyacrylamide gel electrophoresis in the presence of Ca2+, and (c) the affinity of antibodies against mammalian calmodulin for a Paramecium component as demonstrated by both indirect immunofluorescent localization and radioimmunoassay. Indirect immunofluorescence studies reveal that Paramecium calmodulin is distributed in three distinct regions of the cell, i.e., (a) large, spherical cytoplasmic organelles representing perhaps the food vacuoles or other vacuolar inclusions of the cell, (b) along the entire length of oral and somatic cilia, and (c) along a linear punctate pattern corresponding to the kinetics (basal bodies) of the cell.