Regulation of activation state and flagellar wave form in epididymal rat sperm: evidence for the involvement of both Ca2+ and cAMP

Rat sperm from the cauda epididymis exhibit increased motility, longevity, and a distinct circular pattern of flagellar curvature in response to 5 mM procaine-HCl or 0.1 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), reagents that are thought to play a role in the immobilization of free cellular calcium. Triton X-100-extracted sperm models will exhibit the same pattern of motility and curvature as procaine- or TMB-8-activated cells, but only when calcium is removed by a strong chelating agent, and in the presence of cAMP (3 microM). Demembranated sperm models produced from epididymal rat sperm are quiescent unless cAMP is added. In these sperm models, the presence or absence of free calcium mediates a transition in flagellar curvature. The increased activity of the procaine-treated intact cells was not accompanied by a change in cellular ATP content, nor was ATP availability the limiting factor in the quiescent sperm. Therefore, the increased motility produced by procaine is probably mediated by a fall in free intracellular Ca2+ accompanied by a rise in cAMP. Our finding that calcium controls the curvature of sperm flagella may explain altered patterns of flagellar beating, such as the hyperactivated motility that sperm exhibit in the female reproductive tract.     

Regulation of the starfish sperm acrosome reaction by cGMP, pH, cAMP and Ca2+

In the starfish, Asterias amurensis, three components in the jelly coat of eggs, namely acrosome reaction-inducing substance (ARIS), Co-ARIS and asterosap, act in concert on homologous spermatozoa to induce the acrosome reaction (AR). Molecular recognition between the sperm surface molecules and the egg jelly molecules must underlie signal transduction events triggering the AR. Asterosap is a sperm-activating molecule, which stimulates rapid synthesis of intracellular cGMP, pH and Ca(2+). This transient elevation of Ca(2+) level is caused by a K(+)-dependent Na(+)/Ca(2+) exchanger, and the increase of intracellular pH is sufficient for ARIS to induce the AR. The concerted action of ARIS and asterosap could induce elevate intracellular cAMP levels in starfish sperm and the sustained increase in [Ca(2+)], which is essential for the AR. The signaling pathway induced by these factors seems to be synergistically regulated to trigger the AR in starfish sperm.     

Roles of cAMP in regulating microtubule sliding and flagellar bending in demembranated hamster spermatozoa

To understand the mechanism regulating spermatozoa motility, it is important to investigate the mechanism regulating the conversion of microtubule sliding into flagellar bending. Therefore, we analyzed microtubule sliding and its conversion into flagellar bending using a demembranated spermatozoa model in which microtubule sliding and flagellar bending could be analyzed separately by treating the demembranated spermatozoa with and without dithiothreitol, respectively. Using this model, we examined the roles of cAMP and its target molecules in regulating flagellar bending and microtubule sliding. Although flagellar bending did not occur in the absence of cAMP, microtubule extrusion occurred without it, suggesting that cAMP is necessary for the conversion of microtubule sliding into flagellar bending, but not for microtubule sliding itself. The target of cAMP for regulating flagellar bending was not cAMP-dependent protein kinase (PKA), since flagellar bending was still observed in the spermatozoa treated with a PKA-specific inhibitor. Alternatively, the Epac/Rap pathway may be the target. Epac2 and Rap2 were detected in hamster spermatozoa using immunoblotting. Since Rap2 is a GTPase, we investigated the flagellar bending of demembranated spermatozoa treated with GTPgammaS. The treatment markedly increased the beat frequency and bending rate. These results suggest that cAMP activates the Epac/Rap pathway to regulate the conversion of microtubule sliding into flagellar bending.     

Regulation of Na+/Ca2+ exchange activity by cytosolic Ca2+ in transfected Chinese hamster ovary cells

Transfected Chinese hamster ovary cells stably expressing thebovine cardiacNa⁺/Ca²⁺exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca²⁺ concentrations([Ca²⁺]_i)that activateNa⁺/Ca²⁺exchange activity. Ba²⁺ influx wasmeasured in fura 2-loaded, ionomycin-treated cells under conditions inwhich the intracellular Na⁺concentration was clamped with gramicidin at ~20 mM.[Ca²⁺]_iwas varied by preincubating ionomycin-treated cells with either theacetoxymethyl ester of EGTA or medium containing 0-1 mM added CaCl₂. The rate ofBa²⁺ influx increased in asaturable manner with[Ca²⁺]_i,with the half-maximal activation value of 44 nM and a Hill coefficientof 1.6. When identical experiments were carried out with cellsexpressing a Ca²⁺-insensitivemutant of the exchanger, Ba²⁺influx did not vary with[Ca²⁺]_i.The concentration for activation of exchange activity was similar tothat reported for whole cardiac myocytes but approximately an order ofmagnitude lower than that reported for excised, giant patches. Thereason for the difference in Ca²⁺regulation between whole cells and membrane patches is unknown.

     

Bicarbonate actions on flagellar and Ca2+ -channel responses: initial events in sperm activation

At mating, mammalian sperm are diluted in the male and female reproductive fluids, which brings contact with HCO(3)(-) and initiates several cellular responses. We have identified and studied two of the most rapid of these responses. Stop-motion imaging and flagellar waveform analysis show that for mouse epididymal sperm in vitro, the resting flagellar beat frequency is 2-3 Hz at 22-25 degrees C. Local perfusion with HCO(3)(-) produces a robust, reversible acceleration to 7 Hz or more. At 15 mM the action of HCO(3)(-) begins within 5 seconds and is near-maximal by 30 seconds. The half-times of response are 8.8+/-0.2 seconds at 15 mM HCO(3)(-) and 17.5+/-0.4 seconds at 1 mM HCO(3)(-). Removal of external HCO(3)(-) allows a slow return to basal beat frequency over approximately 10 minutes. Increases in beat symmetry accompany the accelerating action of HCO(3)(-). As in our past work, HCO(3)(-) also facilitates opening of voltagegated Ca(2+) channels, increasing the depolarization-evoked rate of rise of intracellular Ca(2+) concentration by more than fivefold. This action also is detectable at 1 mM HCO(3)(-) and occurs with an apparent halftime of approximately 60 seconds at 15 mM HCO(3)(-). The dual actions of HCO(3)(-) respond similarly to pharmacological intervention. Thus, the phosphodiesterase inhibitor IBMX promotes the actions of HCO(3)(-) on flagellar and channel function, and the protein kinase A inhibitor H89 blocks these actions. In addition, a 30 minute incubation with 60 micro M cAMP acetoxylmethyl ester increases flagellar beat frequency to nearly 7 Hz and increases the evoked rates of rise of intracellular Ca(2+) concentration from 17+/-4 to 41+/-6 nM second(-1). However, treatment with several other analogs of cAMP produces only scant evidence of the expected mimicry or blockade of the actions of HCO(3)(-), perhaps as a consequence of limited permeation. Our findings indicate a requirement for cAMP-mediated protein phosphorylation in the enhancement of flagellar and channel functions that HCO(3)(-) produces during sperm activation.     

Hyperactivated motility of bull sperm is triggered at the axoneme by Ca2+ and not cAMP

Hyperactivated motility, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization in vivo. It is characterized by high-amplitude flagellar waves and, usually, highly asymmetrical flagellar beating. It had been suggested, but not tested, that Ca2+ and cAMP switch on hyperactivation by directly affecting the flagellar axoneme. In this study, the direct affects of these agents on the axoneme were tested by using detergent-demembranated bull sperm. As confirmed by TEM, treatment of sperm with 0.2% Triton X-100 disrupted the plasma, acrosomal, and inner mitochondrial membranes, leaving axonemes intact. In the presence of 2 mM ATP, the percentage of reactivated sperm that were hyperactivated increased to 80% when free Ca2+ was increased from 50 to 400 nM. The effect of the Ca2+ in this range was to increase beat asymmetry by increasing the curvature of the principal bend. No additional increases were observed above 400 nM free Ca2+, but motility was suppressed at 1 mM. The ability of Ca2+ to produce hyperactivation depended on ATP availability, such that more ATP was required to produce the high amplitude flagellar bends characteristic of hyperactivated motility than to produce activated motility. Cyclic AMP was not required for reactivation, nor for hyperactivation. Production of hyperactivated motility also required an alkaline environment (pH 7.9-8.5). These results suggest that, provided sufficient ATP is present and pH is sufficiently alkaline, Ca2+ switches on hyperactivation by enabling curvature of the principal bends to increase.     

Regulation of Ca2+ release-activated Ca2+ channels by INAD and Ca2+ influx factor

Thecoupling mechanism between depletion of Ca²⁺ stores in theendoplasmic reticulum and plasma membrane store-operated ion channelsis fundamental to Ca²⁺ signaling in many cell types and hasyet to be completely elucidated. Using Ca²⁺release-activated Ca²⁺ (CRAC) channels in RBL-2H3 cells asa model system, we have shown that CRAC channels are maintained in theclosed state by an inhibitory factor rather than being opened by theinositol 1,4,5-trisphosphate receptor. This inhibitory role can befulfilled by the Drosophila protein INAD (inactivation-noafter potential D). The action of INAD requires Ca²⁺ andcan be reversed by a diffusible Ca²⁺ influx factor. Thusthe coupling between the depletion of Ca²⁺ stores and theactivation of CRAC channels may involve a mammalian homologue of INADand a low-molecular-weight, diffusible store-depletion signal.

     

Regulation of sperm flagellar movement by protein phosphorylation and dephosphorylation

Flagellar motility of Triton models of sea urchin spermatozoa was reactivated by cyclic AMP-dependent protein kinase and a protein factor, termed motility activator, both of which were prepared from the detergent-extract of sea urchin spermatozoa. It was shown that phosphorylation of the motility activator by the protein kinase is necessary for the reactivation of flagellar motility [Ishiguro et al, J. Cell Biol. 92:777-782, 1982; Murofushi et al, in "Biological Functions of Microtubules and Related Structures," Academic Press, 1982]. Reactivating factor was also detected in a KCl-extract of the axoneme fraction devoid of the detergent-extractable materials. The activity of this factor was also cyclic AMP- and protein kinase-dependent. Furthermore, when freshly prepared Triton models were treated with phosphoprotein phosphatase prepared from bovine cardiac muscle, the flagellar motility was drastically suppressed. This inhibition of the motility was partially recovered by the addition of cyclic AMP and protein kinase to the phosphatase-treated models.     

Regulation of sperm flagellar motility by calcium and cAMP-dependent phosphorylation

There is substantial evidence that cAMP-dependent phosphorylation is involved in the activation of motility of spermatozoa as they are released from storage in the male reproductive tract. This evidence includes observations that in vivo activation of motility can be inhibited by protein kinase inhibitors, can be reversed by protein phosphatase treatment of demembranated spermatozoa, and is associated with phosphorylation of sperm proteins, and observations that spermatozoa that have not been activated in vivo can be activated in vitro by cAMP-dependent phosphorylation. Activation in vivo can often be triggered by conditions that increase intracellular pH, but the relevance of this to in vivo activation under natural conditions and the steps between pH increase and cAMP increase have not been fully established. The relationships between changes in the protein substrates for cAMP-dependent phosphorylation and changes in axonemal function are still unknown. Sperm chemotaxis to egg secretions is widespread; in the sea urchin Arbacia, the egg jelly peptide resact has been identified as a chemoattractant. Response to chemoattractants involves changes in asymmetry of flagellar bending waves, and similar changes in asymmetry can be produced in vitro by increases in [Ca++]. Temporal changes in resact receptor occupancy might lead to transient changes in intracellular [Ca++] and the asymmetry of flagellar bending, but many links in this hypothetical sequence remain to be established. Both of these signalling systems offer immediate opportunities for investigations of biochemical pathways leading to easily assayable biological responses. However, complications resulting from interactions between these two systems need to be considered.     

Regulation of the phosphotyrosine content of human sperm proteins by intracellular Ca2+: role of Ca2+-adenosine triphosphatases

An increase in the concentration of intracellular free Ca2+ and in the phosphotyrosine content of specific proteins characterizes human sperm capacitation. Whether tyrosine phosphorylation regulates the intracellular free Ca2+ concentration through modulation of Ca2+-ATPase activity or the phosphotyrosine content is under Ca2+ regulation was investigated using Ca2+-ATPase modulators and tyrosine kinase inhibitors. The presence of the Ca2+-ATPase-inhibitor thapsigargin during human sperm capacitation caused an increase in the cytoplasmic free Ca2+ concentration and was associated with an increase in the phosphotyrosine content of specific sperm proteins. Conversely, a decrease in protein tyrosine phosphorylation was observed when gingerol, a Ca2+-ATPase activator, was present during the incubation period. On the other hand, thapsigargin had no effect on the phosphotyrosine content or the cytoplasmic Ca2+ concentration when spermatozoa were incubated in the presence of the phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX). However, the effect of IBMX on phosphotyrosine-containing proteins appears to be a Ca2+-dependent phenomenon, because it was partly inhibited in spermatozoa pretreated with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM) even though, by itself, BAPTA-AM caused an increase in sperm protein phosphotyrosine content. Tyrosine kinase inhibitors prevented the increase in the phosphotyrosine content without affecting the cytoplasmic free Ca2+ concentration. Based on these findings, the present study suggests that Ca2+-ATPases are involved in the filling of internal Ca2+ stores, such as the acrosome, and are inhibited later during capacitation. Their inhibition allows an increase in cytoplasmic free Ca2+, which is involved in the subsequent increase in the phosphotyrosine content of specific sperm proteins.     

Modulation of the asymmetry of sea urchin sperm flagellar bending by calmodulin

Sea urchin spermatozoa demembranated with Triton X-100 in the presence of EGTA, termed potentially asymmetric, generate asymmetric bending waves in reactivation solutions containing EGTA. After they are converted to the potentially symmetric condition by extraction with Triton and millimolar Ca++, they generate symmetric bending waves in reactivation solutions containing EGTA. In the presence of EGTA, their asymmetry can be restored by addition of brain calmodulin or the concentrated supernatant obtained from extraction with Triton and millimolar Ca++. These extracts contain calmodulin, as assayed by gel electrophoresis, radioimmunoassay, activation of brain phosphodiesterase, and Ca++-dependent binding of asymmetry-restoring activity to a trifluorophenothiazine-affinity resin. Conversion to the potentially symmetric condition can also be achieved with trifluoperazine substituted for Triton during the exposure to millimolar Ca++, which suggests that the calmodulin-binding activity of Triton is important for this conversion. These observations suggest that the conversion to the potentially symmetric condition is the result of removal of some of the axonemal calmodulin and provide additional evidence for axonemal calmodulin as a mediator of the effect of Ca++ on the asymmetry of flagellar bending.     

Uncoupling of Chlamydomonas flagellar gene expression and outgrowth from flagellar excision by manipulation of Ca2+

Chlamydomonas cells respond to certain environmental stimuli by shedding their flagella. Flagellar loss induces a rapid, transient increase in expression of a specific set of genes encoding flagellar proteins, and assembly of a new flagellar pair. While flagellar gene expression and initiation of flagellar outgrowth are normally tightly coupled to flagellar excision, our results demonstrate that these processes can be uncoupled by manipulating Ca2+ levels or calmodulin activity. In our experiments, wild-type cells were stimulated to excise their flagella using mechanical shearing, and at times after deflagellation, flagellar lengths were measured and flagellar mRNA abundance changes were determined by S1 nuclease protection analysis. When extracellular Ca2+ was lowered by addition of EGTA to cultures before excision, flagellar mRNA abundance changes and flagellar outgrowth were temporally uncoupled from flagellar excision. When extracellular Ca2+ was lowered immediately after excision or when calmodulin activity was inhibited with W-7, flagellar outgrowth was uncoupled from flagellar excision and flagellar mRNA abundance changes. Whenever events in the process of flagellar regeneration were temporally uncoupled, the magnitude of the flagellar mRNA abundance change was reduced. These results suggest that flagellar gene expression may be regulated by multiple signals generated from these events, and implicate Ca2+ as a factor in the mechanisms controlling flagellar regeneration.     

Regulation of Ca2+ transport by sarcoplasmic reticulum Ca2+-ATPase at limiting [Ca2+

The factors regulating Ca2+ transport by isolated sarcoplasmic reticulum (SR) vesicles have been studied using the fluorescent indicator Fluo-3 to monitor extravesicular free [Ca2+]. ATP, in the presence of 5 mM oxalate, which clamps intravesicular [Ca2+] at approximately 10 microM, induced a rapid decline in Fluo-3 fluorescence to reach a limiting steady state level. This corresponds to a residual medium [Ca2+] of 100 to 200 nM, and has been defined as [Ca2+]lim, whilst thermodynamic considerations predict a level of less than 1 nM. This value is similar to that measured in intact muscle with Ca2+ fluophores, where it is presumed that sarcoplasmic free [Ca2+] is a balance between pump and leaks. Fluorescence of Fluo-3 at [Ca2+]lim was decreased 70% to 80% by histidine, imidazole and cysteine. The K0.5 value for histidine was 3 mM, suggesting that residual [Ca2+]lim fluorescence is due to Zn2+. The level of Zn2+ in preparations of SR vesicles, measured by atomic absorption, was 0.47+/-0.04 nmol/mg, corresponding to 0.1 mol per mol Ca-ATPase. This is in agreement with findings of Papp et al. (Arch. Biochem. Biophys., 243 (1985) 254-263). Histidine, 20 mM, included in the buffer, gave a corrected value for [Ca2+]lim of 49+/-1.8 nM, which is still higher than predicted on thermodynamic grounds. A possible 'pump/leak' mechanism was tested by the effects of varying active Ca2+ transport 1 to 2 orders with temperature and pH. [Ca2+]lim remained relatively constant under these conditions. Alternate substrates acetyl phosphate and p-NPP gave similar [Ca2+]lim levels even though the latter substrate supported transport 500-fold slower than with ATP. In fact, [Ca2+]lim was lower with 10 mM p-NPP than with 5 mM ATP. The magnitude of passive efflux from Ca-oxalate loaded SR during the steady state of [Ca2+]lim was estimated by the unidirectional flux of 45Ca2+, and directly, following depletion of ATP, by measuring release of 40Ca2+, and was 0.02% of Vmax. Constant infusion of CaCl2 at [Ca2+]lim resulted in a new steady state, in which active transport into SR vesicles balances the infusion rate. Varying infusion rates allows determination of [Ca2+]-dependence of transport in the absence of chelating agents. Parameters of non-linear regression were Vmax=853 nmol/min per mg, K0.5(Ca)=279 nM, and nH(Ca)=1.89. Since conditions employed in this study are similar to those in the sarcoplasm of relaxed muscle, it is suggested that histidine, added to media in studies of intracellular Ca2+ transients, and in the relaxed state, will minimise contribution of Zn2+ to fluophore fluorescence, since it occurs at levels predicted in this study to cause significant overestimation of cytoplasmic free [Ca2+] in the relaxed state. Similar precautions may apply to non-muscle cells as well. This study also suggests that [Ca2+]lim in the resting state is a characteristic feature of Ca2+ pump function, rather than a balance between active transport and passive leakage pathways.     

Sperm-activating peptide induces asymmetric flagellar bending in sea urchin sperm

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, induces various sperm responses including a transient increase in the intracellular Ca2+ concentration. However, it has not been clarified how speract modulates sperm motility and whether it functions as a chemoattractant. To confirm the effect of speract on sperm motility, we observed the flagellar bending response to speract in sperm of Hemicentrotus pulcherrimus, in experiments using caged speract and a lighting system for a microscope newly developed with a power LED. We found that speract induces increases in curvature of swimming paths and changes flagellar bending shape to asymmetric. These facts show that speract directly regulates flagellar motility, and suggest that speract-induced increases in intracellular Ca2+ concentration play an actual role in regulation of the flagellar movement.     

Regulation of leukocyte locomotion by Ca2+

Neutrophils migrate towards sites of inflammation and infection by chemotaxis. Their motility is dependent on the actin cytoskeleton and on adhesion to extracellular substrates, but how these are regulated in response to stimuli is not clear. This review focuses on the potential role of Ca(2+) as a second messenger in neutrophil motility. Several effects of Ca(2+) and Ca(2+)-binding proteins on the stability and crosslinking of actin polymers have been demonstrated in vitro. Nevertheless, the complex mechanism by which Ca(2+) regulates actin in neutrophils is not fully understood. In addition, intracellular Ca(2+) regulates the intergin-mediated adhesion of neutrophils to extracellular matrix.     

Analysis of the flagellar bending waves of ejaculated ram sperm

The variability of flagellar movement, illustrated by the highly heterogeneous nature of the ejaculated sperm population of the ram, was analyzed by the use of a stroboscopic technique and an adapted microphotographic 24 X 36 camera system. The multiple-moving-exposures (MME) records give very distinct successive sequences of the flagellar beats and are particularly suitable for the analysis of bend development and propagation along the tail. With this technique, the parameters of the flagellar bending waves of ejaculated ram sperm have been determined. Most of the sperm have planar flagellar beatings; few are rolling under the conditions of observation. The trajectories of the gametes are mostly linear; nevertheless, some have circular paths. The analysis of bending has been focused on two examples for which the difference in the progressiveness ratio was maximum. The circular pathways for ram spermatozoa are linked to an asymmetry between principal and reverse bend probably induced by differences in wave propagation evidenced along the flagellum. A typical sperm flagellar movement may be related either to the conditions of the observations or to some differences in the maturation process of the sperm.     

Regulation of cardiac sarcolemmal Ca2+ channels and Ca2+ transporters by thyroid hormone

In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca²⁺-channels, Na⁺–Ca²⁺ exchange and Ca²⁺-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca²⁺-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca²⁺ and Ca²⁺ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca²⁺ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [³H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (B_max) without any change in the dissociation constant for receptor-ligand complex (K_d) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake and ouabain-sensitive Na⁺–K⁺ ATPase were decreased whereas the Mg²⁺-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na⁺–K⁺ ATPase activity, whereas Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake, and Mg²⁺-ATPase activities were unchanged. The V_max and K_a for Ca²⁺ of cardiac sarcolemmal Na⁺–Ca²⁺ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca²⁺-transport processes is regulated by thyroid hormones and the modification of Ca²⁺-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart.     

Effects of Mg2+ and Ca2+ on photoinduced Euglena flagellar responses

The flagellar frequency and waveform of Euglena were analyzed under full illumination (420-700 nm) and in a restricted wavelength band (530- 700 nm) when the cells were in a medium containing Mg2+ or had been microinjected with Mg2+, Mn2+, or Ca2+ in solution. Magnesium abolished the change in flagellar frequency and the reversal in waveform that cells exhibit when illuminated by a 530-700 nm wavelength band. Under this restricted illumination, Ca2+ caused an increase in flagellar waveform reversal and a decrease in beating frequency. The flagellar motility of cells impaled on a microelectrode was examined in cells illuminated with various wavelengths.