Flicker in erythrocytes. I. Theoretical models and registration techniques

The phenomenon of stochastic low-frequency oscillations of erythrocyte cell membrane, termed usually the flicker of erythrocytes, is reviewed. The first part describes the theoretical models of erythrocyte flickering and the registration techniques. The relations are given and analyzed which connect the shape of both the frequency and the spatial spectra of stochastic membrane oscillations with geometrical and mechanical parameters of the erythrocyte and with the ambient physical characteristics. The existing concepts of excitation mechanisms of the membrane flickering are presented.     

Luminal fluid tonicity regulates airway ciliary beating by altering membrane stretch and intracellular calcium

The coordinated, directional beating of airway cilia drives airway mucociliary clearance. Here we explore the hypothesis that airway surface liquid osmolarity is a key regulator of ciliary beating. Cilia in freshly isolated human and murine airways visualized with streaming video-microscopy exhibited a reciprocal dependence on a physiological range of luminal fluid osmolarities, across the entire range of ciliary activity (0-20 beats per sec). Increasing osmolarity slowed or completely abrogated, while lower osmolarity dramatically stimulated ciliary beating. In parallel, epithelial cell height and importantly, intracellular calcium levels (as judged by fluorescence imaging) also changed. Moreover, ciliary beating was stimulated by isosmotic solutions containing membrane permeant osmolytes, suggesting that cell size and membrane stretch (governed by apical fluid tonicity), rather than osmolarity itself, contribute to the activation. These findings shed light on the pathophysiology of diseases of mucociliary clearance such as cystic fibrosis and other chronic inflammatory lung diseases.     

Identification of methyl resonances in the 1H NMR spectrum of incubated blood cell lysates

The origin of low frequency methyl resonances which appear in the spin-echo 1H nuclear magnetic resonance spectra of incubated blood cell lysates was investigated by several techniques including 1H and 13C nuclear magnetic resonance spectroscopy, electrophoresis, high performance liquid chromatography, gel filtration, and amino acid analysis. These resonances were identified as arising from methyl moieties of leucine and valine. Other peaks which also appeared in the spectra of incubated blood cell lysates were assigned to methyl groups of alanine and threonine. The free amino acids are products of neutral proteases located on the leukocyte membrane or able to act on the extracellular medium. Since more than one enzyme appears to be implicated, it is possible that both membrane and granule proteases take part in the hydrolysis. Comparison of rates of product formation in white cell lysates incubated with human serum albumin, and with red cell lysate, suggests that erythrocyte peptidases also contribute to proteolysis in the latter case.     

Rapid local oscillations of the surface of the human erythrocyte

The transverse displacements of the human erythrocyte surface with amplitude 300-400 nm in the frequency range 0.2-30 Hz are recorded on the minimal area erythrocyte rim (approximately 0.5 X 0.5 microns). These local oscillations of the surface are diminished at hypoosmotic erythrocyte swelling, on addition of substances which increase the membrane rigidity (0.01% glutaraldehyde, 0.5 mM 4-hydroxymercuribenzoate, cell membrane stain--0.002% Heliogen Blue) and on discocyte--echinocyte transformation due to addition of 1-2 mM 2,4-dinitrophenol. The amplitude of transverse displacements is reduced by 1.7-2 times on erythrocytes of patients with inherent microspherocytosis. These erythrocytes have inherent defects in spectrin. It is suggested that spectrin is important for rapid local oscillations of the human erythrocyte surface.     

Flicker in erythrocytes. II. Results of experimental studies

The phenomenon of stochastic low-frequency oscillations of erythrocyte cell membrane, termed usually the flicker of erythrocytes, is reviewed. The first part [Biol. Membrany (Rus.), 2009, vol. 26, no. 5, pp. 352–369] describes theoretical models of erythrocyte flickering and the registration techniques. In the second part presented below the main experimental results are reviewed, the problem of identification of acting mechanisms of flicker excitation is analyzed, and flicker interrelations are considered with the membrane functioning as well as with the dynamics of proteins embedded in the membrane. The possibilities and the prospects of medical diagnostics applications of flicker of erythrocytes are discussed briefly.     

Effect of cholesterol on human erythrocyte membrane. A spin label study

The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids. 1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents. 2. On increasing the cholesterol or ASl content in the cell membrane, the spin label was gradually immobilized. 3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes. 4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterol-lipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed.     

Interaction of the antivirus agents remantadine and amantadine with lipid membranes and the influence on the curvature of human red cells

Surface potential difference, conductance, and elasticity changes of bilayer lipid membranes induced by the antivirus drugs amantadine and remantadine were measured. An influence on the human erythrocyte shape was shown. Both drugs are stomatocytogenic. The adsorption at the cytoplasmatic membrane was electrophoretically proved. The heat-induced vesiculation is partly inhibited. No microvesicles were observed. Instead, large tails which did not detach from the cell body were seen. The general conclusion is that these amphiphilic adamantane derivatives are membrane agents which modify membrane interaction processes, possibly by influencing the bending properties.     

Regulation of Membrane Flexibility in Human Erythrocytes.

We have used spin-labels to detect prostaglandin E induced changes in erythrocyte membranes. The observed changes in spin-label resonance spectra can be mimicked in erythrocyte ghosts by loading them with cAMP or cGMP. These changes can also be observed by adding either of these cyclic nucleotides to intact cells. This entry of cyclic nucleotides into intact cells is blocked by an inhibitor of the anion channel. We suggest that the observed changes in paramagnetic resonance spectra are due to changes in lipid "fluidity" that are brought about by changes in the biochemical state of membrane-associated proteins (such as spectrin) and in the direct or indirect biophysical interactions of these proteins with membrane lipids.     

Endothelin depolarizes membrane voltage and increases intracellular calcium concentration in human ciliary muscle cells

The ciliary muscle which is involved in accommodation and regulation of aqueous humour outflow resistance resembles smooth muscle in other parts of the body. In the present investigation we used an established primary cell line (H7CM) to study the effects of endothelin, a novel vasoconstrictor peptide, on membrane voltage (V) and intracellular calcium in cultured human ciliary muscle cells. Membrane voltage was measured in confluent monolayers of H7CM cells using conventional microelectrodes. Intracellular calcium concentration [( Ca]i) was measured in single H7CM cells using the fluorescent calcium indicator fura-2. Under resting conditions V averaged -66.9 +/- 0.7 mV (mean +/- SEM, n = 125). Endothelin (10(-10)-10(-6)M) induced a dose-dependent reversible membrane voltage depolarization and a dose-dependent rise in [Ca]i. The initial calcium peak was followed by a recovery phase during which oscillations of [Ca]i occurred. The initial calcium peak was not dependent on the presence of extracellular calcium and was not abolished in the presence of the calcium antagonist verapamil (10(-4)M). Thus it is probably mediated by a release of calcium from intracellular reservoirs. We conclude that cultured human ciliary muscle cells express a functional endothelin receptor.     

Electron spin resonance studies of an animal model of human congenital myotonia: increased erythrocyte membrane fluidity in rats with 20,25-diazacholesterol-induced myotonia.

Electron spin resonance experiments have been performed on erythrocyte membranes from rats with myotonia induced by treatment with 20,25-diazacholesterol. The results suggest that erythrocyte membranes in this animal model of human congenital myotonia possess a highly significantly increased surface membrane fluidity compared to that of controls. Alterations in the physical state of membrane proteins were not apparent. These findings, also present in human congenital myotonia [Butterfield, Chesnut, Roses & Appel, 1976, Nature (London) 263:159; Butterfield, 1977 (Submitted for publication)], strengthen the concepts that increased membrane fluidity is associated with the presence of myotonia and that congenital myotonia may be a diffuse membrane disease.     

Parameter estimation with bio-inspired meta-heuristic optimization: modeling the dynamics of endocytosis

Background

Ciliary dysfunction leads to a number of human pathologies, including primary ciliary dyskinesia, nephronophthisis, situs inversus pathology or infertility. The mechanism of cilia beating regulation is complex and despite extensive experimental characterization remains poorly understood. We develop a detailed systems model for calcium, membrane potential and cyclic nucleotide-dependent ciliary motility regulation.

Results

The model describes the intimate relationship between calcium and potassium ionic concentrations inside and outside of cilia with membrane voltage and, for the first time, describes a novel type of ciliary excitability which plays the major role in ciliary movement regulation. Our model describes a mechanism that allows ciliary excitation to be robust over a wide physiological range of extracellular ionic concentrations. The model predicts the existence of several dynamic modes of ciliary regulation, such as the generation of intraciliary Ca²⁺ spike with amplitude proportional to the degree of membrane depolarization, the ability to maintain stable oscillations, monostable multivibrator regimes, all of which are initiated by variability in ionic concentrations that translate into altered membrane voltage.

Conclusions

Computational investigation of the model offers several new insights into the underlying molecular mechanisms of ciliary pathologies. According to our analysis, the reported dynamic regulatory modes can be a physiological reaction to alterations in the extracellular environment. However, modification of the dynamic modes, as a result of genetic mutations or environmental conditions, can cause a life threatening pathology.     

Functional activity of ciliated outgrowths from cultured human nasal and tracheal epithelia

Primary cultures of respiratory epithelium were produced as outgrowths from human fetal and adult tracheal and nasal polyp explants. Video recordings of the epithelial cell outgrowths were carried out after 5 days of culture and the ciliary beating frequency was analyzed by using a video technique. Uniform fields of differentiated ciliated cells were observed near the edge of the explant. In the transition region of the outgrowth from the explant to the outgrowth periphery, isolated ciliated cells were present, as well as cells with fused cilia. The ciliary beating frequency of the outgrowth of well-differentiated ciliated cells (13.5 +/- 1.4 Hz) was significantly higher (p less than 0.001) than the beating frequency of both the explant (11.9 +/- 0.7 Hz) and the ciliated cells with fused cilia (9.8 +/- 1.7 Hz). The same differentiation stages and functional activities were observed in the outgrowth cultures, whatever their origin. These in vitro models are comparable with each other and therefore could be useful for studying the ciliogenesis and functional activity of the human respiratory epithelium.     

Video-microscopy observations of fast dynamic processes in the protozoon Giardia lamblia

Video-microscopy in combination with digital image processing was used to analyze dynamic processes associated to the life cycle of Giardia lamblia trophozoites. These parasites swim and attach to the epithelial cells, producing the disease known as Giardiasis. Giardia is a multiflagellar cell, presenting 4 pairs of flagella. With the use of analogue and digital tools, we observed that in cells attached to glass slides only 2 of the 4 pairs present active beating (wave propagation). The frequency observed was 17-18 Hz to the anterior and 8-11 Hz to the ventral flagella. These data resulted from several hours of recording using both analogue video and high-speed digital camera. The caudal pair did not show active beating patterns and the same holds true for the posterior one. In this latter pair, oscillations were observed, but they were always associated to the transit of the wave produced by the ventral pair. The analysis performed with free moving cells showed that during its forward dislocation, Giardia lamblia presented either a lateral rocking or a complete rotational (tumbling) movement around its longitudinal axis. A dislocation of the caudal region of the cell both in the lateral and dorso-ventral direction was observed. This movement was completely independent from the flagellar beating and it is likely to be produced by a microtubular complex located in the caudal portion of the cell. The adhesion process of Giardia lamblia was also followed by video-microscopy and the data showed that the ventral disk had an active participation in this process.     

Electron spin resonance studies of an animal model of human congenital myotonia: Increased erythrocyte membrane fluidity in rats with 20,25-diazacholesterol-induced myotonia

Summary Electron spin resonance experiments have been performed on erythrocyte membranes from rats with myotonia induced by treatment with 20,25-diazacholesterol. The results suggest that erythrocyte membranes in this animal model of human congenital myotonia possess a highly significantly increased surface membrane fluidity compared to that of controls. Alterations in the physical state of membrane proteins were not apparent. These findings, also present in human congenital myotonia [Butterfield, Chesnut, Roses & Appel, 1976,Nature (London)263:159; Butterfield, 1977 (Submitted for publication)], strengthen the concepts that increased membrane fluidity is associated with the presence of myotonia and that congenital myotonia may be a diffuse membrane disease.     

Extracellular ATP induces hyperpolarization and motility stimulation of ciliary cells.

Cellular membrane potential and ciliary motility were examined in tissues cultures prepared from frog palate and esophagus epithelia. Addition of micromolar concentrations of extracellular ATP caused membrane hyperpolarization and enhanced the beat frequency. These two effects of ATP were 1) dose dependent, reaching a maximum at 10 microM ATP; 2) dependent on the presence of extracellular Ca2+ or Mg2+; 3) insensitive to inhibitors of voltage-gated calcium channels; 4) abolished after depleting the intracellular Ca2+ stores with thapsigargin; 5) attenuated by quinidine (1 mM), Cs+ (5-20 mM), and replacement of extracellular Na+ by K+; 6) insensitive to charybdotoxin (5-20 nM), TEA (1-20 microM), and apamin (0.1-1 microM); 7) independent of initial membrane potential; and 8) unaffected by amiloride. In addition, extracellular ATP induced an appreciable rise in intracellular Ca2+. Addition of thapsigargin caused an initial enhancement of the ciliary beat frequency and membrane hyperpolarization. These results strongly suggest the involvement of calcium-dependent potassium channels in the response to ATP. The results show that moderate hyperpolarization is closely associated with a sustained enhancement of ciliary beating by extracellular ATP.     

Effect of Chlamydia trachomatis infection on ciliary activity in single cells from cultures of human nasal polyps

We have tested the hypothesis that the ciliary activity of epithelial cells from human nasal polyps is altered after infection with Chlamydia trachomatis. Ciliated epithelial cells from human nasal polyps were cultured and infected with C. trachomatis. The measurement of ciliary beating was based on a technique which enables one to monitor a fraction of a single ciliated cell. A marked decrease of ciliary beating frequency versus time was observed 24 h after infection with C. trachomatis. About 50% of the cilia of infected cells were paralysed 48 h post-infection. The potential effect of C. trachomatis infection on the physiological functions dependent on cilia is discussed.     

Calcium oscillations in the olfactory nonsensory cells of the goldfish, Carassius auratus

Background

The olfactory nonsensory cells contribute to the maintenance of normal functions of the olfactory epithelium (OE). Specifically, the ciliated nonsensory cells of teleosts play important roles in the odorant detection by OE in aqueous environment. Their cilia show strong beating activities and cause water flow at the OE surface, making the detection of odorants by OE more efficient. Because intracellular Ca²⁺ level has been reported to play an important role in ciliary beating, the ciliary beating activity may be regulated by intracellular Ca²⁺ dynamics of these ciliated nonsensory cells.

Methods

We performed Ca²⁺ imaging experiments to analyze the Ca²⁺ dynamics in acutely dissociated OE cells of the goldfish. Furthermore, we examined the contribution of the Ca²⁺ dynamics to the ciliary beating frequency (CBF) at the surface of the intact OE.

Results

Olfactory nonsensory cells showed both spontaneous intracellular Ca²⁺ oscillations and propagating intercellular Ca²⁺ waves. Application of 2-aminoethoxydiphenylborate (2-APB), which antagonizes IP₃-induced Ca²⁺ release from intracellular stores suppressed these Ca²⁺ oscillations. Furthermore, 2-APB application to the intact OE lamellae resulted in the decrease of CBF at the surface of the OE.

Conclusions

These results indicate that spontaneous intracellular calcium oscillations persistently up-regulate the ciliary beating at the surface of the OE in teleosts.

General significance

Ciliary beating activity at the surface of OE can be regulated by the Ca²⁺ dynamics of olfactory nonsensory cells. Because this ciliary movement causes inflow of external fluid into the nostril, this regulation is suggested to influence the efficiency of odorant detection by OE.     

Organization of actin microfilaments in the apical border of oviduct ciliated cells

Actin microfilaments were localized in quail oviduct ciliated cells using decoration with myosin subfragment S1 and immunogold labeling. These polarized epithelial cells show a well developed cytoskeleton due to the presence of numerous cilia and microvilli at their apical pole. Most S1-decorated microfilaments extend from the microvilli downward towards the upper part of the ciliary striated rootlets with which they are connected. From the microvillous roots, a few microfilaments connect the proximal part of the basal body or the basal foot associated with the basal body. Microfilament polarity is shown by S1 arrowheads pointing away from the microvillous tip to the cell body. Furthermore, short microfilaments are attached to the plasma membrane at the anchoring sites of basal bodies and run along the basal body. The polarity of these short microfilaments is directed from the basal body anchoring fibers downward to the cytoplasm. At the cell periphery, microfilaments from microvillous roots and ciliary apparatus are connected with those of the circumferential actin belt which is associated with the apical zonula adhaerens. Together with the other cytoskeletal elements, the microfilaments increase ciliary anchorage and could be involved in the coordination of ciliary beating. Moreover, microvilli surrounding the cilia probably modify ciliary beating by offering resistance to cilium bending. The presence of microvilli could explain the fact that mainly the upper part of the cilia appanars to be involved in the axonemal bending in metazoan ciliated cells.     

Distinct axonemal processes underlie spontaneous and stimulated airway ciliary activity

Cilia are small organelles protruding from the cell surface that beat synchronously, producing biological transport. Despite intense research for over a century, the mechanisms underlying ciliary beating are still not well understood. Even the nature of the cytosolic molecules required for spontaneous and stimulated beating is debatable. In an effort to resolve fundamental questions related to cilia beating, we developed a method that integrates the whole-cell mode of the patch-clamp technique with ciliary beat frequency measurements on a single cell. This method enables to control the composition of the intracellular solution while the cilia remain intact, thus providing a unique tool to simultaneously investigate the biochemical and physiological mechanism of ciliary beating. Thus far, we investigated whether the spontaneous and stimulated states of cilia beating are controlled by the same intracellular molecular mechanisms. It was found that: (a) MgATP was sufficient to support spontaneous beating. (b) Ca(2+) alone or Ca(2+)-calmodulin at concentrations as high as 1 microM could not alter ciliary beating. (c) In the absence of Ca(2+), cyclic nucleotides produced a moderate rise in ciliary beating while in the presence of Ca(2+) robust enhancement was observed. These results suggest that the axonemal machinery can function in at least two different modes.