Chemotaxis as a mechanism of the locomotor activity of olfactory cilia

This article is devoted to the modern understanding of the locomotor activity of olfactory cilia as a form of chemotaxis. It includes an analysis of published data and the results of experimental research that was performed by the authors.     

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.     

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.     

The proteome of rat olfactory sensory cilia

Olfactory sensory neurons expose to the inhaled air chemosensory cilia which bind odorants and operate as transduction organelles. Odorant receptors in the ciliary membrane activate a transduction cascade which uses cAMP and Ca²⁺ for sensory signaling in the ciliary lumen. Although the canonical transduction pathway is well established, molecular components for more complex aspects of sensory transduction, like adaptation, regulation, and termination of the receptor response have not been systematically identified. Moreover, open questions in olfactory physiology include how the cilia exchange solutes with the surrounding mucus, assemble their highly polarized set of proteins, and cope with noxious substances in the ambient air. A specific ciliary proteome would promote research efforts in all of these fields. We have improved a method to detach cilia from rat olfactory sensory neurons and have isolated a preparation specifically enriched in ciliary membrane proteins. Using LC‐ESI‐MS/MS analysis, we identified 377 proteins which constitute the olfactory cilia proteome. These proteins represent a comprehensive data set for olfactory research since more than 80% can be attributed to the characteristic functions of olfactory sensory neurons and their cilia: signal processing, protein targeting, neurogenesis, solute transport, and cytoprotection. Organellar proteomics thus yielded decisive information about the diverse physiological functions of a sensory organelle.     

Clustering of cyclic-nucleotide-gated channels in olfactory cilia

Olfactory cilia contain the known components of olfactory signal transduction, including a high density of cyclic-nucleotide-gated (CNG) channels. CNG channels play an important role in mediating odor detection. The channels are activated by cAMP, which is formed by a G-protein-coupled transduction cascade. Frog olfactory cilia are 25-200 microm in length, so the spatial distribution of CNG channels along the length should be important in determining the sensitivity of odor detection. We have recorded from excised cilia and modeled diffusion of cAMP into a cilium to determine the spatial distribution of the CNG channels along the ciliary length. The proximal segment, which in frog is the first 20% of the cilium, appears to express a small fraction of the CNG channels, whereas the distal segment contains the majority, mostly clustered in one region.     

Single unit recording from olfactory cilia.

Sensory cilia from olfactory receptor cells can be pulled into a patch pipette located above the mucus layer of an olfactory mucosa. While the pipette does not form a tight electrical seal with the ciliary membrane, it nevertheless allows to record current transients driven by action potentials arising in the olfactory neuron. This method is an alternative to single-unit-recording with electrodes pushed into the mucosa and, in some respects, to patch clamp recordings from isolated olfactory cells. Its advantage is technical simplicity and minimal disturbance of the neuron from which signals are derived. Less than 5% of the chemosensitive apical surface of the neuron is covered by the pipette. The neuron remains in situ and its cilia remain covered with some mucus. (However, mucus is in part dissolved by the bathing solution). Odorant thresholds in the picomolar range were thus obtained.     

Large olfactory responses of the carp after complete removal of olfactory cilia

To study the role of olfactory cilia on olfactory reception, the carp olfactory cilia were removed by modified "ethanol-calcium shock" and the bulbar responses were recorded before and after deciliation. Large olfactory responses to various amino acids were observed after complete deciliation. The relation between magnitude of olfactory response and alanine concentration before and after deciliation was essentially unchanged. The present results suggests that the olfactory cilia may not be necessary for receptor neuron function in the carp.     

Mechanism of olfactory masking in the sensory cilia

Olfactory masking has been used to erase the unpleasant sensation in human cultures for a long period of history. Here, we show a positive correlation between the human masking and the odorant suppression of the transduction current through the cyclic nucleotide–gated (CNG) and Ca²⁺-activated Cl⁻ (Cl_(Ca)) channels. Channels in the olfactory cilia were activated with the cytoplasmic photolysis of caged compounds, and their sensitiveness to odorant suppression was measured with the whole cell patch clamp. When 16 different types of chemicals were applied to cells, cyclic AMP (cAMP)-induced responses (a mixture of CNG and Cl_(Ca) currents) were suppressed widely with these substances, but with different sensitivities. Using the same chemicals, in parallel, we measured human olfactory masking with 6-rate scoring tests and saw a correlation coefficient of 0.81 with the channel block. Ringer''s solution that was just preexposed to the odorant-containing air affected the cAMP-induced current of the single cell, suggesting that odorant suppression occurs after the evaporation and air/water partition of the odorant chemicals at the olfactory mucus. To investigate the contribution of Cl_(Ca), the current was exclusively activated by using the ultraviolet photolysis of caged Ca, DM-nitrophen. With chemical stimuli, it was confirmed that Cl_(Ca) channels were less sensitive to the odorant suppression. It is interpreted, however, that in the natural odorant response the Cl_(Ca) is affected by the reduction of Ca²⁺ influx through the CNG channels as a secondary effect. Because the signal transmission between CNG and Cl_(Ca) channels includes nonlinear signal-boosting process, CNG channel blockage leads to an amplified reduction in the net current. In addition, we mapped the distribution of the Cl_(Ca) channel in living olfactory single cilium using a submicron local [Ca²⁺]_i elevation with the laser photolysis. Cl_(Ca) channels are expressed broadly along the cilia. We conclude that odorants regulate CNG level to express masking, and Cl_(Ca) in the cilia carries out the signal amplification and reduction evenly spanning the entire cilia. The present findings may serve possible molecular architectures to design effective masking agents, targeting olfactory manipulation at the nano-scale ciliary membrane.     

Guanylate cyclase in olfactory cilia from rat and pig

A guanylate cyclase was identified in cilia from rat and pig olfactory epithelia. Enzyme activities were 200-250 and 90-100 pmol/min.mg-1, respectively. Activity required the presence of non-ionic detergents, e.g., 0.1% Lubrol PX. MnGTP, not MgGTP was used as a substrate. Furthermore, 0.9 mM free Mn2+ was necessary for optimal activity indicating a regulatory site for a divalent cation. The guanylate cyclase displayed sigmoidal Michaelis-Menten kinetics suggesting cooperativity between MnGTP and enzyme. S0.5 was 160 microM MnGTP. The Hill coefficient of 1.7 indicates that more than one class of substrate-binding sites interact in a positive cooperative manner. ATP inhibited the enzyme and linearized plots of substrate kinetics with MnGTP. SH-Blocking agents reversibly inhibited enzyme activity. Sodium azide and nitroprusside were without effect as were several odorants. A guanylate cyclase activity in cilia from tracheal tissue had properties similar to the olfactory enzyme.     

Odorant- and guanine nucleotide-stimulated phosphoinositide turnover in olfactory cilia

Isolated olfactory cilia from the channel catfish (Ictalurus punctatus) exhibited phosphatidylinositol-4,5-bisphosphate phosphodiesterase (E.C.3.1.4.11) activity. The phosphodiesterase activity was stimulated in the presence of an odorant for the catfish, namely the amino acid L-alanine. The enzyme activity was also stimulated in the presence of GTP and its nonhydrolyzable analogues. The activation of the phosphodiesterase by guanine nucleotides, in combination with the identification of guanine nucleotide-binding protein(s) in the isolated cilia, indicate the probable participation of a guanine nucleotide-binding protein in stimulation of phosphoinositide turnover in the olfactory receptor neuron.     

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.     

Mathematical modeling of movement of cilia in olfactory cells

A mathematical model of the movement of olfactory cilia in different conditions was constructed. The realization of the model includes the development of a mechanical mathematical rheological model of the behavior of a continuous deformable medium, the development of the method of solution adapted to this problem, and obtaining a numerical solution, which takes into account different starting data. The mathematical modeling of the dynamic behavior of a deformable medium was performed using a system of equations for the dynamics of the deformable medium and the solution of the corresponding nonstationary system of equations in partial derivatives.     

Dynamics of 'immotile' olfactory cilia in the silkmoth Antheraea

Living olfactory sensory dendrites of the silkmoth Antheraea which are modified cilia lacking the central microtubule pair have been observed by means of video microscopy in sensilla from which the apical tips had been pinched off as well as in vitro after isolation. Dendrites project out of the opened hair tips cither spontaneously without manipulation or after application of basal pressure via a syringe connected to the haemolymph side. Spontaneously appearing dendrites can repeatedly project up to ca. 60 mum from, and retract back into, the hairs. They tend to remain straight, but curve if they project too far and bend on meeting an obstacle. The average elongation velocity of the dendrites is 0.4 mum/sec. After application of basal pressure, large numbers of dendrites immediately slide out of the apically opened hairs. These dendrites usually detach at their bases and float free in the solution until settling down at the bottom of the petri dish. They are able to make active movements, for example bending between points of attachment. Dendrites tend to adhere to other dendrites, sometimes making sliding movements against each other. The ciliary olfactory dendrites are backed by a large number of microtubules which appear to be interconnected by fine filaments, most probably microtubule-associated proteins (MAPs). The elongation and shortening of the dendrites is explained here by a sliding-filament mechanism similar to the one acting in 'true motile' cilia. As the cytoskcleton is not as highly organized as in the latter, the resulting movements are limited to elongation and contraction, bending being brought about only passively by apical resistance. Membrane beads have been observed to appear on, and move along, the dendrites. Their number increases with the age of the preparation.     

Spatial distribution of calcium-gated chloride channels in olfactory cilia

Background

In vertebrate olfactory receptor neurons, sensory cilia transduce odor stimuli into changes in neuronal membrane potential. The voltage changes are primarily caused by the sequential openings of two types of channel: a cyclic-nucleotide-gated (CNG) cationic channel and a calcium-gated chloride channel. In frog, the cilia are 25 to 200 µm in length, so the spatial distributions of the channels may be an important determinant of odor sensitivity.

Principal Findings

To determine the spatial distribution of the chloride channels, we recorded from single cilia as calcium was allowed to diffuse down the length of the cilium and activate the channels. A computational model of this experiment allowed an estimate of the spatial distribution of the chloride channels. On average, the channels were concentrated in a narrow band centered at a distance of 29% of the ciliary length, measured from the base of the cilium. This matches the location of the CNG channels determined previously. This non-uniform distribution of transduction proteins is consistent with similar findings in other cilia.

Conclusions

On average, the two types of olfactory transduction channel are concentrated in the same region of the cilium. This may contribute to the efficient detection of weak stimuli.     

Bovine olfactory cilia preparation: thiol-modulated odorant-sensitive adenylyl cyclase

We have characterized the adenylyl cyclase activity in a newly developed preparation of isolated olfactory cilia from the bovine chemosensory neuroepithelium. Like its counterparts from frog and rat, the ciliary enzyme was stimulated by guanine nucleotides, by forskolin, and by a variety of odorants in the presence of GTP. The main difference between the bovine olfactory cilia preparation and the frog and rat olfactory cilia preparation is that odorant stimulation of the bovine olfactory adenylyl cyclase is strongly inhibited by submillimolar concentrations of dithiothreitol. This inhibition is a consequence of a concomitant increase in the GTP-stimulated level and the decrease of the odorant stimulation of the enzyme. Nasal respiratory cilia have a much lower level of adenylyl cyclase activity and show no odorant stimulation. Owing to the large quantities of material available, the bovine olfactory cilia preparation is advantageous for studies of the proteins involved in chemosensory transduction.     

The control of anthozoan cilia by the basal apparatus

The ciliary basal apparatus in the pharynx of the sea anemone, Calliactis parasitica (Couch), is composed of two centrioles, a single striated rootlet at least 20 microns long, and a basal foot, to the tip of which is attached a bundle of microtubules leading to the rootlet. When the basal apparatus is sectioned in the plane of the ciliary power-stroke, the distal centriole, with which the cilium base is continuous, is rarely found to be erect. The orientation of the distal centriole is determined by bending in the basal apparatus. Bending occurs only in the plane of the ciliary power-stroke towards the side from which the basal foot projects, and it is closely correlated with membrane buckling in the belt desmosome region of the cell apex. Associated with the belt desmosome, but not directly with the basal apparatus, are bundles of filaments. These filaments are of two size classes, 5-6 and 10 nm in diameter. A model is presented in which the 5-6 nm filaments form the basis of a contractile system which mediates membrane buckling in the region of the belt desmosome. This action effectively shortens the cell apex and thus forces the apparatus to bend. The precise reorientation of the distal centriole is a result of the mechanical properties of the basal apparatus.     

Formation and maturation of olfactory cilia monitored by odorant receptor-specific antibodies

The responsiveness of olfactory sensory neurons (OSNs) is based on odorant receptors (ORs) residing in the membrane of chemosensory cilia. It is still elusive as to when and how olfactory cilia are equipped with OR proteins rendering them responsive to odorants. To monitor the appearance of OR proteins in sensory compartments of OSNs, the olfactory epithelium of mice at various stages of prenatal development (lasting 19 days from conception) was investigated using immunohistochemical approaches and antibodies specific for different OR subtypes. These experiments uncovered that OR proteins accumulated in dendritic knobs of OSNs before the initiation of ciliogenesis (embryonic stage E12). As the first cilia were formed (E13), immunostaining in the knobs diminished. Cilia extended uprightly into the nasal cavity and were immunoreactive along the entire length, and particularly intense labeling was observed in expanded tips of cilia. During this phase of development (up to E18), the number of cilia per knob continuously increased. In the course of perinatal stages, longer cilia began to bend off and lie flat on the epithelial surface. The multiple cilia of a knob extended in length, and eventually the ciliary meshwork reached the characteristic complex pattern. In all stages, OR immunostaining was visible along the entire cilium. Thus, OR-specific antibodies allowed, for the first time, monitoring at the level of light microscopy the generation, outgrowth, and maturation of cilia in OSNs.     

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.