In mammals, Ca2+ influx as L-type Ca2+ current (ICa) triggers the release of Ca2+ from sarcoplasmic reticulum (SR) and Ca2+-induced Ca2+ release (CICR) is critical for excitation-contraction coupling. In fish, the relative contribution of external and internal Ca2+ is unclear. Here, we characterized the role of ICa to trigger SR Ca2+ release in rainbow trout ventricular myocytes using ICa regulation by Ca2+ as an index of CICR. ICa was recorded with a slow (EGTA) or fast (BAPTA) Ca2+ chelator in control and isoproterenol conditions. In the absence of β-adrenergic stimulation, the rate of ICa inactivation was not significantly different in EGTA and BAPTA (27.1 ± 1.8 vs. 30.3 ± 2.4 ms), whereas with isoproterenol (1 μM), inactivation was significantly faster with EGTA (11.6 ± 1.7 vs. 27.3 ± 1.6 ms). When barium was the charge carrier, inactivation was significantly slower in both conditions . Quantification revealed that without isoproterenol, only 39% of ICa inactivation was due to Ca2+, while with isoproterenol, inactivation was Ca2+-dependent (∼65%) and highly reliant on SR Ca2+ (∼46%). Thus, SR Ca2+ is not released in basal conditions, and ICa is the main trigger of contraction, whereas during a stress response, SR Ca2+ is an important source of cytosolic Ca2+. This was not attributed to differences in SR Ca2+ load because caffeine-induced transients were not different in both conditions. Therefore, Ca2+ stored in SR of trout cardiomyocytes may act as a safety mechanism, allowing greater contraction when higher contractility is required, such as stress or exercise.Cardiomyocyte contraction depends on rapid changes in intracellular Ca The resulting net increase in global systolic Ca2+ transient, formed by the spatial and temporal sum of those local Ca2+ events, subsequently activates the contractile machinery within the cardiomyocyte   from cloomyocyte . An asso2+ entry during each heartbeat and prevent cell Ca2+ overload, LTCCs undergo inactivation, induced by both Ca2+ and voltage  (2+ (2+ load and modulates the AP duration (2+ channels includes Ca2+-dependent facilitation (CDF) that potentiates LTCC Ca2+ influx during repeated activity and contributes to increasing the force-frequency relationship of some mammalian hearts during exercise  or whether internal Ca2+, stored in the SR, plays an active role , it is possible to discriminate between action of CICR from the SR upon ICa or not. This approach was first developed in neurons to characterize neurotransmitter secretion , and the Ba2+ current via LTCC (IBa) is used as a measure of VDI (Oncorhyncus mykiss) as an experimental model to 1) quantify ICa inactivation and 2) investigate whether ICa can trigger SR Ca2+ release. In addition, we have recorded caffeine-induced systolic Ca2+ transients to ascertain the presence of Ca2+ in the SR of fish cardiomyocytes.One approach to characterize the role of SR Caecretion . In cardvents it . To furte of VDI . Thus, wICa does not induce SR Ca2+ release and that external Ca2+ is the main trigger of cardiac contraction. In contrast, during β-adrenergic stimulation, CICR occurs and Ca2+ released from the SR is a major source of Ca2+ flux into the cytosol and of the intracellular Ca2+ transient that activates rainbow trout cardiomyocytes contraction. We propose that the Ca2+ stored in the SR of rainbow trout myocytes may act as a reserve and is released only when extra Ca2+ is required for greater cell contraction to maintain or enhance cardiac function, such as during stress.We show that in control conditions Oncorhyncus mykiss) were obtained from Chirk Fish Farm . Fish were held in freshwater tanks at 12 ± 1°C with a 12:12-h light-dark cycle and fed with commercial fish pellets. All procedures were in accordance with local animal handling protocols and adhere to UK Home Office legislation.Rainbow trout (2+-free isolation solution (see Solutions). After 10 min, the isolation solution was supplemented with collagenase , trypsin , and BSA (0.7 mg/ml), and the perfusion was continued for another 15 min. Next, the heart was cut below the bulbus and the ventricle was separated from the atrium. The ventricle was cut in pieces, and individual cells were released by gently agitating the muscle pieces. Cell suspension was filtered and kept in isolation solution at 12 ± 1°C in a water bath to prolong cell viability and were used within 8 h. Only elongated cells (spindle shape) with clear cross-striations and without granulation or blebs were used for experiments. All experiments were performed at room temperature (20 ± 1°C).Cardiomyocytes were isolated by enzymatic dissociation, as previously described , 55. RaiAll the experiments described in this article were recorded using the whole cell configuration of the patch-clamp technique settings and properties, as described previously . An Axop+- and K+-free internal and external solutions to avoid contamination by overlapping ionic currents and to allow the use of a physiological holding potential .Experiments were performed using Naotential . ICa was25; 50: APD50; 90: APD90).APs were evoked by 5-ms subthreshold current steps. Trains of pulses were applied at 0.1 and 1 Hz. AP duration (APD) was measured as the duration from the overshoot to three different percentages of repolarization  for 10 min at room temperature. Fura-2 AM fluorescence was elicited by alternate (every 2 ms) illumination with 340 and 380 nm light obtained using a monochromator  in front of a Xenon excitation lamp. The fluorescence emitted at 510 nm was monitored using a photomultiplier tube (Cairn Research). The ratio  was used as an index of [Ca2+]i. Cells were superfused with control solution (see Solutions) and electrically field-stimulated at 0.33 Hz via a pair of platinum electrodes until steady state. Rapid application of caffeine (20 mM) was used to assess SR Ca2+ content. pClamp software (Axon Instruments) was used for recordings and analysis.Cardiomyocytes were incubated with the CaICa was measured as the difference between the peak of the inward current and the current at the end of the depolarizing pulse. Because the decay of ICa varied between experimental conditions, the kinetics of ICa inactivation were characterized as the time required for the current to decay to 0.37 of the peak amplitude (T0.37), as previously described  was performed as previously described (ICa (pA/ms) during the 250-ms test pulse to obtain total Ca2+ influx during the pulse.escribed . Therefoescribed  and expl2PO4, 4 MgSO4, 50 taurine, 20 glucose, and 10 HEPES (pH to 6.9 with NaOH). The control bathing solution (Ringer) used for action potential and Ca2+ cycling recording contained (in mM): 150 NaCl, 5.4 KCl, 1.5 MgSO4, 0.4 NaH2PO4, 2 CaCl2, 10 glucose, and 10 HEPES (pH to 7.7 with NaOH). For ICa recording, cells were locally perfused with Na+ and K+-free solution, which contained (in mM): 137 TEACl, 6 CsCl, 1 MgCl2, 20 HEPES, 10 glucose, 2 CaCl2 set to pH 7.6 with TEAOH. In some experiments, CaCl2 was replaced with BaCl2. The pipette solution for measurement of ICa contained (in mM): 10 TEACl, 125 CsCl, 1 MgCl2, 5 Mg-ATP, 2 EGTA, 1 CaCl2, 10 HEPES and 0.3 GTPTris (pH to 7.2 with CsOH). In some experiments, EGTA was substituted to 10 mM BAPTA. The free Ca2+ concentration in the presence of EGTA is 186 nM and BAPTA is 25 nM  × 100}, we estimated that total CDI represents 39% of ICa inactivation in control conditions. To separate SR Ca2+ release-induced CDI from total CDI, we further compared ICa recorded in EGTA and BAPTA. Thus, the difference between IR20EGTA and IR20BAPTA represents the current inactivated by SR-induced CDI. By normalizing to total CDI {[(IR20BAPTA − IR20EGTA)/(IR20Ba − IR20EGTA)] × 100}, we estimated that SR CDI represents 16% of total CDI. Collectively, these data show that under basal conditions, VDI is the major determinant of ICa inactivation in rainbow trout ventricular cardiomyocytes. This differs from mammalian species , where ICa and the Ca2+ release from the SR (measured as IR20). ICa and IR20 in control conditions  and during perfusion with 1 μM isoproterenol . Under control conditions (ICa recorded with 2 mM EGTA in pipette solution), ICa density is low and inactivation is slow (IR20 elevated), indicating an absence of Ca2+ release. Indeed, IR20 decreases linearly as ICa density increases, most probably due to an increase of Ca2+ entry via LTCCs, which, in turn, inactivate the channel (see introduction). Perfusion with 1 μM isoproterenol increased ICa density and ICa inactivation (IR20). IR20 plateaued at a value (∼0.2), and the relation between ICa density and ICa inactivation is now flat because CICR occurs. A threshold of ∼6 pA/pF is needed for this  will clamp the bulk of cytosolic Ca2+, while allowing the concentration of Ca2+ in the restricted diffusion space to change, hence permitting ICa to trigger SR Ca2+ release .In the present study, we have applied a similar method to that previously described in mammalian cardiomyocytes  and usedsee Ref. . Applica2+ chelator (10 mM BAPTA), this local Ca2+ cycling is prevented (no CICR triggered), such that ICa inactivation is slower (2+ concentration close to the mouths of channels due to Ca2+ entering via the LTCC only (SR Ca2+ release is prevented)  . Thus, bcoupling .2+ buffer and NCX block [important given the relative role in trout myocytes  CICR does not occur and 2) ICa is sufficient to elevate [Ca2+]i enough to trigger contraction of rainbow trout cardiomyocytes. These observations validate the current model of most fish cardiac EC coupling suggested in previous studies. They had been mainly attributed to the small dimensions of the fish cardiomyocyte that facilitate the activation of the myofilaments by sarcolemmal Ca2+ and also to the limited effect of the SR inhibitor ryanodine on cell contraction .In addition, it was previously demonstrated that both temperature (acute change and acclimation) and heart rate play a key role in the sensitivity to ryanodine in the rainbow trout myocardium . In agre2+ in the SR, since we observed substantial caffeine-evoked Ca2+ transients of magnitude, previously described in rainbow trout cardiomyocytes .Importantly, we show that when the CaVornanen  suggeste2+ released by the SR depends on the amplitude of ICa, the trigger for release, and the SR Ca2+ load . Therefore, our data demonstrate that it is possible to induce CICR in fish cardiomyocytes if ICa is of sufficient amplitude  and, importantly, that SR CDI accounts for nearly half of total CDI (46% due to SR CDI). Interestingly, the proportion of ICa inactivation due to total CDI and SR CDI observed during β-adrenergic stimulation in rainbow trout cardiomyocytes was similar to inactivation of ICa in mammalian species observed under control conditions [e.g., in rat . This idea is supported by the observation that a low concentration of caffeine was sufficient to induce CICR, even with low amplitude of the trigger, ICa (above). In mammals, basal ICa density is >6 pA/pF. Therefore, it is tempting to speculate that SR Ca2+ release is triggered regardless of the current density  and The Biotechnology and Biological Sciences Research Council .No conflicts of interest, financial or otherwise, are declared by the authors.Author contributions: C.C. and F.B. conception and design of research; C.C., L.S., D.E.W., and F.B. performed experiments; C.C., L.S., and F.B. analyzed data; C.C., L.S., H.A.S., and F.B. interpreted results of experiments; C.C. and F.B. prepared figures; C.C. drafted manuscript; C.C., L.S., D.E.W., H.A.S., and F.B. edited and revised manuscript; C.C., L.S., D.E.W., H.A.S., and F.B. approved final version of manuscript.


Ionocytes within the adult gills or larval skin are critical sites for transcellular Ca2+ uptake in teleosts. The ionocytes of zebrafish were found to contain transcellular Ca2+ transporters, epithelial Ca2+ channel (ECaC), plasma membrane Ca2+-ATPase 2 (PMCA2), and Na+/Ca2+ exchanger 1b (NCX1b), providing information about the molecular mechanism of transcellular Ca2+ transports mediated by ionocytes in fish. However, more evidence is required to establish whether or not a similar mechanism of transcellular Ca2+ transport also exists in others teleosts. In the present study, ecac, pmca2, and ncx1 were found to be expressed in the branchial ionocytes of tilapia, thereby providing further support for the mechanism of transcellular Ca2+ transport through ionocytes previously proposed for zebrafish. In addition, we also reveal that low Ca2+ water treatment of tilapia stimulates Ca2+ uptake and expression of ecac and cyp11b (the latter encodes a cortisol-synthesis enzyme). Treatment of tilapia with exogenous cortisol (20 mg/l) enhanced both Ca2+ influx and ecac expression. Therefore, increased cyp11b expression is suggested to enhance Ca2+ uptake capacity in tilapia exposed to low Ca2+ water. Furthermore, the application of cortisol receptor antagonists revealed that cortisol may regulate Ca2+ uptake through glucocorticoid and/or mineralocorticoid receptor (GR and/or MR) in tilapia. Taken together, the data suggest that cortisol may activate GR and/or MR to execute its hypercalcemic action by stimulating ecac expression in tilapia.Ca Due to the hypercalcemic action of cortisol . In cell lines transfected with teleost glucocorticoid receptor (GR) or mineralocorticoid receptor (MR), cortisol treatment activated the transcription of a glucocorticoid response element (GRE)-element containing plasmid  ECaC, PMCA2, and NCX1 are responsible for transcellular epithelial Ca2+ transport in tilapia, and (2) cortisol acts via GR and/or MR to regulate Ca2+ uptake by modulating expression of these Ca2+ transporters in tilapia. To test these hypotheses, we designed experiments to answer the following specific questions: (1) are ecac, pmca2, and/or ncx1 expressed in ionocytes in tilapia? (2) Does the external Ca2+ level regulate ecac, pmca2, and ncx1 expression in tilapia? (3) Does cortisol modulate Ca2+ uptake and the mRNA expression of ecac, pmca2, and ncx1 in tilapia? And finally, (4) does cortisol regulate Ca2+ uptake through the GR and/or MR?The purpose of the present study is to enhance our comprehensive understanding of fish CaOreochromis mossambicus), 1–50 g in body weight, were taken from stocks at the Institute of Cellular and Organismic Biology, Academia Sinica, and kept in freshwater  at 27 °C under a 14 h:10 h light:dark photoperiod. Tilapia larvae were acquired as follows: fertilized eggs were collected from the mouths of female tilapia and incubated in aerated FW. Fertilized eggs that hatched at the same time were used in the experiments. All experiments were conducted on yolk-sac larvae, and no feeding occurred. The incubation water was changed daily to control water quality. For sampling, fish (adult and hatched embryos) were anesthetized with buffered MS-222  and then dissected. Sampling was performed in accordance with the guidelines of the Academia Sinica Institutional Animal Care and Utilization Committee .Tilapia  supplemented with adequate CaSO4·2H2O, MgSO4·7H2O, NaCl, K2HPO4, and KH2PO4. The Ca2+ concentrations of the high- and low-Ca2+ media were 2 and 0.02 mM, respectively, but all other ion concentrations in all media were the same as those in local tap water . Variations in ion concentrations were maintained within 10 % of the predicted values by monitoring with an atomic absorption spectrophotometer . For acclimation, hatched embryos and adults were incubated with high- and low-Ca2+ media for 3 days and 2 weeks, respectively. Fish were sampled for the assay at the end of the acclimation period.Artificial fresh waters with high (2 mM) and low (0.02 mM) CaCortisol dosages were selected with reference to previous studies , appropriate amounts of tilapia tissues or embryos were collected. For the RNA extraction, the samples were homogenized in 1 ml Trizol reagent  and then referred to manufacturer’s protocol. Finally, the quantity and quality of total RNA were assessed based on the absorbance at 260 nm and the ratio of the absorbance at 260 and 280 nm, as measured using a Nanodrop ND-2000 .18, 5 mM dithiothreitol, and 200 units PowerScript reverse transcriptase  for 1.5 h at 42 °C, followed by a 15 min incubation at 70 °C. For PCR amplification, 2 μl cDNA was used as template in a 50 μl final reaction volume containing 0.25 mM dNTP, 2.5 units EX-Taq polymerase , and 0.2 μM of each primer. GenBank accession numbers of the sequences for primer sets were used as follows: ecac, GenBank BankIt Submission ID:1884659; pmca2, AAK15034; ncx1, AY283779; gadph, FN673690.The mRNA was purified from the total RNA extracted from tilapia tissues with a commercial kit . For cDNA synthesis, 0.36 μg of mRNA was reverse transcribed in a final volume of 20 μl containing 0.5 mM dNTPs, 2.5 μM oligo (dT)ecac (342 bp fragment), forward 5′-AGAGGATGAAAAGGAAACGG-3′, reverse 5′-ATGGCATAATACTGCGGAAA-3′; ncx1 (310 bp fragment), forward 5′-TGCCGTCTACCACTACACCC-3′, reverse 5′-GCAGCGACCTAAAATCCAAC-3′; pmca2 (693 bp fragment), forward 5′-AACAACCTGGTGCGTCA-3′, reverse 5′-GGGGTCCTCTATTCCGA-3′; gadph (415 bp fragment), forward 5′-AATACGACCCCTCCTCCAT-3′, reverse 5′-TACCCCAGCACTCCTTTCA-3′. The amplicons were all sequenced to ensure that the PCR products were the desired gene fragments.The primer sets used for Reverse transcription-PCR analysis were as follows: ecac, pmca2, and ncx1 were obtained by PCR and inserted into a pGEM-T easy vector . After linearization by restriction enzyme digestion, the plasmids were subjected to in vitro transcription with T7 and SP6 RNA polymerase  to produce sense and anti-sense transcripts, respectively. Dig-labeled RNA probes were examined with RNA gels and dot-blot assay to confirm the quality and concentration.PCR fragments of tilapia l-lysine coated slides . After brief washing with PBST, slides were incubated with hybridization buffer (HyB) containing 50 % formamide, 5× SSC, and 0.1 % tween-20 for 5 min at 65 °C. Prehybridization was performed for 2 h at 65 °C with HyB+ . For hybridization, samples were incubated with100 ng RNA probe in 200 μl HyB+ at 65 °C overnight. Next, the slides were washed at 65 °C for 10 min in 75 % HyB and 25 % 2× saline sodium sitrate (SSC), 10 min in 50 % HyB and 50 % 2× SSC, 10 min in 25 % HyB and 75 % 2× SSC, 10 min in 2× SSC, and finally 30 min in 0.2× SSC at 70 °C . Further washes were performed at room temperature for 5 min in 75 % 0.2× SSC and 25 % phosphate buffered saline with 0.1 % triton X-100 (PBST), 5 min in 50 % 0.2× SSC and 50 % PBST, 5 min in 25 % 0.2× SSC and 75 % PBST, and 5 min in PBST. After the series of washes, slides were incubated for 2 h in blocking solution containing 5 % sheep serum and 2 mg/ml bovine serum albumin (BSA) in PBST, and then incubated with 1:2500 antibody  in blocking solution for another 2 h at room temperature. Finally, sections were washed with PBST plus blocking reagent, and were then transferred to staining buffer. The staining reaction was performed with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and p-nitroblue tetrazolium chloride (NBT) in staining buffer until the signal was strong enough for analysis.Excised gills were fixed with 4 % paraformaldehyde for 3 h at 4 °C and then washed several times with phosphate buffered saline (PBS). Fixed samples were immersed in PBS containing 30 % sucrose overnight, and embedded in OCT compound embedding medium  at −20 °C. Frozen cross-sections of 10 μm were cut with a CM 1900 rapid sectioning cryostat  and attached to poly-+/K+-ATPase (2.5 µg/ml in PBS) at 4 °C overnight. Samples were washed in PBS for 30 min twice, and then incubated with goat anti-mouse IgG conjugated with FITC  for 1 h at room temperature. Images were acquired with a Leica TCS-NT confocal laser scanning microscope .Sections were washed several times with PBST after in situ hybridization. Blocking was performed in 3 % BSA at room temperature for 2 h, and sections were then incubated with α5 mouse anti-Nacyp11b (160 bp fragment), forward 5′-ACATCTTCAGTCATGCGGAG-3′, reverse 5′-ATGAGGTCCAAAGATAGCTGC-3′; mr (145 bp fragment), forward 5′-GCTGTGGAAGGTCAGCATAA-3′, reverse 5′-ACTTCTTGGATTTCCGTGCT-3′; gr (159 bp fragment), forward 5′-AAAGGCCAGCACAACTACCT-3′, reverse 5′-CTGGACACCCTTTAACCGAT-3′; ecac (152 bp fragment), forward 5′-CTGTCTCTGGCCTCGACTT-3′, reverse 5′- CCTCCGTTTCCTTTTCATCCT-3′; pmca2(157 bp fragment), forward 5′-TCTGTCAGGAAGTCGATGA-3′, reverse 5′-CCTTGTCTCGTGGACGGAA-3′; ncx1(261 bp fragment), forward 5′-CAAGAGAGCCACCCATGATATCTT-3′, reverse 5′-TTTCCGTCTCACCGGGTTT-3′; for β-actin (132 bp fragment), forward 5′-GGTGGGTATGGGTCAGAAAG-3′, reverse 5′-TGCCAGATCTTCTCCATGTC-3′’. The β-actin was used a house-keeping gene to normalize mRNA expression and its expression was not modulated in the experiments of Ca2+ and pharmacological treatment in this study.The mRNA purification and cDNA synthesis procedures are described above in the section for RT-PCR. Q-PCR reactions were performed with an ABI7000 sequence detection system  in a final volume of 20 μl containing 10 μl 2× SYBR Green master mix , 100 nM primer pairs, and 8 μl cDNA. The standard curve of each gene was checked in the linear range with GAPDH as an internal control. The primer sets used for Q-PCR were as follows: 2+ influx was performed as described previously  were collected from the tracer media at 0.5 and 2.5 h after transfer. Counting solution  was added to the samples, and the radio activities were counted with a liquid scintillation β-counter . Ca2+ influx rates were calculated using the following formula:Qi and Qf (cpm ml−1) refer to initial (0.5 h) and final (2.5 h) radioactivities in the tracer media, Vi and Vf (ml) refer to initial and final volumes of the tracer media, SAi and SAf are initial and final specific activities (cpm mmole−1), t (2 h) is incubation time, and W (g) is fish body weight.Measurement of Cap < 0.05). Data are presented as the mean ± SD and were analyzed by one-way analysis of variance (ANOVA) and Student’s t test.Group datasets were confirmed to be normally distributed by Anderson–Darling Normality test  antibody, revealing that ecac, pmca2, and ncx1b are expressed in the ionocytes of gill filaments  or high (2.0 mM) Cater Fig. a. Furthents Fig. b.Fig. 3E2+ levels on tilapia larvae were examined by acclimating hatching tilapia embryos to low (0.02 mM) or high (2.0 mM) Ca2+ water for 3 days. After 3 days, larvae were sampled for analysis of Ca2+ influx and gene expression. Similar to the adult, tilapia larvae acclimated to low Ca2+ water also exhibited significantly upregulated Ca2+ influx and ecac expression  for 3 days. After the treatment period, larvae were sampled to analyze Ca2+ influx and transporter expression. We report that treatment with exogenous cortisol (10 and 20 mg/l) resulted in significant stimulation of Ca2+ influx and ecac expression, but did not modulate expression of pmca2 or ncx1b  also clearly enhanced ecac expression in tilapia larvae . We report that treatment with either GR or MR antagonist dramatically decreases the stimulatory effect of exogenous cortisol on ecac transcription  Na+, (3) Ca2+, and (4) K+ and acid–base, respectively  than that of pmca2 and ncx1 in tilapia  in zebrafish  was specifically detected in branchial ionocytes and exposure to low Ca2+ water resulted in significant stimulation of both Ca2+ influx and ecac expression in tilapia, similar to the previous findings in zebrafish  (Liao et al. 2+ homeostasis.For summary, the mRNA expression of three Ca


Pseudomonas aeruginosa is a facultative human pathogen, and a major cause of nosocomial infections and severe chronic infections in endocarditis and in cystic fibrosis (CF) patients. Calcium (Ca2+) accumulates in pulmonary fluids of CF patients, and plays a role in the hyperinflamatory response to bacterial infection. Earlier we showed that P. aeruginosa responds to increased Ca2+ levels, primarily through the increased production of secreted virulence factors. Here we describe the role of putative Ca2+-binding protein, with an EF-hand domain, PA4107 (EfhP), in this response. Deletion mutations of efhP were generated in P. aeruginosa strain PAO1 and CF pulmonary isolate, strain FRD1. The lack of EfhP abolished the ability of P. aeruginosa PAO1 to maintain intracellular Ca2+ homeostasis. Quantitative high-resolution 2D-PAGE showed that the efhP deletion also affected the proteomes of both strains during growth with added Ca2+. The greatest proteome effects occurred when the pulmonary isolate was cultured in biofilms. Among the proteins that were significantly less abundant or absent in the mutant strains were proteins involved in iron acquisition, biosynthesis of pyocyanin, proteases, and stress response proteins. In support, the phenotypic responses of FRD1 ΔefhP showed that the mutant strain lost its ability to produce pyocyanin, developed less biofilm, and had decreased resistance to oxidative stress (H2O2) when cultured at high [Ca2+]. Furthermore, the mutant strain was unable to produce alginate when grown at high [Ca2+] and no iron. The effect of the ΔefhP mutations on virulence was determined in a lettuce model of infection. Growth of wild-type P. aeruginosa strains at high [Ca2+] causes an increased area of disease. In contrast, the lack of efhP prevented this Ca2+-induced increase in the diseased zone. The results indicate that EfhP is important for Ca2+ homeostasis and virulence of P. aeruginosa when it encounters host environments with high [Ca2+]. Pseudomonas aeruginosa is a facultative pathogen and a leading cause of severe nosocomial infections in both immunocompetent and immunocompromised patients P. aeruginosa is one of the primary organisms that forms biofilms on airway mucosal epithelium of patients with cystic fibrosis (CF) where it contributes to airway blockage and cellular damage. P. aeruginosa also causes infective endocarditis and device-related infections with high morbidity and mortality rates P. aeruginosa biofilm infections are increasingly difficult to treat with traditional antibiotic therapy, and are often not eradicated by host defensive processes 2+) is a well-known signaling molecule that regulates a number of essential processes in eukaryotes 2+ homeostasis have been implicated in many human diseases, including diseases associated with bacterial infections, such as CF pulmonary infections and endocarditis. Ca2+ metabolism is recognized to be central to the pathology of CF 2+ is a part of a hyperinflamatory host response to bacterial infection, and accumulates in airway epithelia, pulmonary and nasal liquids of CF patients 2+ also plays a significant role in the physiology of certain bacteria, affecting maintenance of cell structure, motility, chemotaxis, cell division and differentiation, gene expression, transport, and spore formation P. aeruginosa have been shown to maintain intracellular Ca2+ at sub-micromolar levels and produce Ca2+ transients in response to environmental and physiological factors 2+ regulation in prokaryotes are not well defined.Calcium (Ca2+ are carried out by Ca2+-binding proteins (CaBPs), which may function as Ca2+ sensors, signal transducers, Ca2+ buffers or Ca2+-stabilized proteins. Prokaryotic genomes also encode CaBPs with different Ca2+-binding motifs, including the EF-hand motif 2+-binding loop flanked by two α-helices. Acidic amino acids in the loop are preferentially bound by Ca2+2+ - induced conformational changes required for function 2+ transducer calsymin CasA from Rhizobium etli2+ buffers and stabilizers calerythrin from Saccharopolyspora erythraeaStreptomyces coelicolor, and Escherichia coli transglycosylase MltB 2+- binding motifs are classified as hypothetical proteins with unknown physiological functions.In eukaryotes, the regulatory effects of Ca2+ modulates proteome profiles of P. aeruginosa, influences biofilm architecture, and alters production of several secreted virulence factors P. aeruginosa hypothetical protein, PA4107, contains EF-hand Ca2+-binding motifs. We also identified PA4107 as having sequence similarity within the EF-hand motif to CasA of R. etli. Here, we further analyzed PA4107, which we designate EfhP (EF hand protein) and hypothesized that it plays roles in P. aeruginosa response to Ca2+. We generated deletion mutants lacking efhP in P. aeruginosa strain PAO1 and in the CF clinical isolate, FRD1, and studied their Ca2+-dependent phenotypes by global quantitative proteomics. Since certain stress response proteins had reduced abundances in the proteomes of the efhP deletion strains, we assayed the mutants for resistance to chemical and oxidative stresses. We also characterized the effect of the mutation on pyocyanin production and survival at low iron. Finally, we tested Ca2+- induced virulence of the mutant and wild-type strains in a plant infection model. The results indicate that EfhP is involved in plant virulence and in Ca2+- enhanced adaptation of P. aeruginosa to host environments.Previously, we showed that CaPseudomonas aeruginosa FRD1 and PAO1 were used in this study. P. aeruginosa FRD1 is an alginate-overproducing (mucoid) CF pulmonary isolate P. aeruginosa PAO1 is the non-mucoid strain used for the original genome sequencing study 4, 0.15 mM NaH2PO4, 0.34 mM K2HPO4, and 145 mM NaCl, 20 µl trace metals, 1 ml vitamin solution. Trace metal solution (per liter of 0.83 M HCl): 5.0 g CuSO4.5H2O, 5.0 g ZnSO4.7H2O, 5.0 g FeSO4.7H2O, 2.0 g MnCl2.4H2O). Vitamins solution (per liter): 0.5 g thiamine, 1 mg biotin. The pH of the medium was adjusted to 7.0. The level of Ca2+ in BMM was below the detection level when measured by QuantiChrom™ calcium assay kit. CaCl2.2H2O when used was added to final concentration 5 or 10 mM. For experiments with no iron, trace metal solution contained no FeSO4.7H2O, and the corresponding BMM was referred to as no iron BMM (BMM-NI). The level of total Fe in BMM-NI was below the detection level when measured by Ferrozine assay 600 of 0.3, and used to inoculate 100 ml of BMM medium in 250-ml flasks. Growth data are based on at least 3 biological replicates. Biofilm formation was quantified using abiotic solid surface assay in 96-well plates as described previously For quantitative growth assays, cultures were pre-cultured in 5 ml tubes to mid-log phase, diluted to obtain optical density ODPseudomonas genome database Rhizobium etli as the query The putative EF-hand protein was first identified from the efhP) was deleted from the chromosomes of PAO1 and FRD1 and replaced with the gentamicin omega fragment using the allelic exchange strategy as described previously 5′-CCCGGGAGCGCTCCTTGATCGGCGGGC-3′), EfhPBamH1Rev (5′-ACGGATCCGAGCAGGCTGGCGGAAGTCTTTTG-3′), EfhPBamH1For (5′-GCGGATCCAGCACTGAGCCTTTCCACAC-3′), and EfhPEco47Rev (5′-GCGTCGAGCGCTCCGGGCGCACTCCGCG-3′). The deletions mutants were complemented in trans by ligating the efhP PCR product into the XbaI restriction site, behind the Ptrc promoter of pMF36 efhP PCR product were: PA4107 Xba 5′ (5′-TCTAGACCGGCCGCGTTTACTGTGGAG-3′) and PA4107 Xba 3′(5′-TCTAGAGGGCGCCGCCAGGGTGTGG-3′). The resulting plasmid, pMF470, was introduced into the mutant strains by triparental mating using the pRK2013 conjugation helper plasmid.PA4107 . Iodacetamide and 3-[(3-cholamidopropyl) dimethylamonio]-1-propanesulfonate (CHAPS) were from Acros Organics  and MP Biomedicals . All chemicals were of analytical grade.Cells from planktonic and biofilm cultures were obtained as described previously 2+ versus their matched counterparts cultivated without added Ca2+. Proteins of interest were excised from the gels and identified based on peptide mass fingerprint analyzed on 4700 MALDI-TOF/TOF Mass spectrometer , using the Applied Biosystems GPS (version 3.6) software, MASCOT search engine and NCBInr database. A protein was considered identified if the hit fulfilled four criteria: (1) it was statistically significant (a MASCOT search score above 75), (2) the number of the matched peptides was at least five, (3) the protein sequence coverage was above 20%, and (4) the predicted molecular mass and pI were consistent with the experimentally determined values.The gels were imaged, and the digital images were analyzed by using Progenesis Workstation Software . A minimum of two biological replicates were performed for each condition, and the signal intensity of each spot was averaged over the replicates. The signal intensities (volumes) of protein spots were normalized against total signal intensity detected on a gel . For protein identification we targeted proteins that met two criteria: (1) the protein had a normalized volume (NV) greater than 0.03, and (2) the proteins were at least threefold more or less abundant in the planktonic or biofilm proteomes cultivated with 10 mM CaP. aeruginosa (100 µl) were grown on BMM agar for 24 h, and collected using 3–5 ml of saline. The samples were extracted with 3 ml of chloroform followed by extraction with 1 ml of 0.2 N HCl. The absorbance was measured at 520 nm, and pyocyanin concentrations (µg/ml) were calculated by multiplying the absorbance by extinction coefficient 17.072 P. aeruginosa were grown on BMM-NI agar plates for 24 h. To deprive the cells of iron, seven passages onto fresh BMM-NI agar were performed. The cells were collected using 3–5 ml of saline. Alginate was precipitated with 2% cetyl pyridium chloride then isopropanol, dissolved in saline, and detected by using the modified carbazole method described in For pyocyanin analysis, chloroform extraction of the pigment followed by a spectrophotometric assay was used as described in 2O2 for 1 h or heat shock at 50°C for 30 min. The concentrations and the duration of treatments were optimized to reach approximately 50% cell survival in wild type FRD1. Prior to exposure, the cultures were grown to mid-log phase in BMM containing 10 mM CaCl2 or no added Ca2+, then diluted to obtain optical density of 0.2 at 600 nm. Viable cells were determined after serial dilution as colony form units (CFUs) before and after exposure. Results are expressed as the mean survival percentages from three independent experiments.The sensitivity of cells to stress was tested by exposing cultures to 1% ethanol, 10% DMSO, 1 mM H4. P. aeruginosa wild type and mutant strains were incubated for 18 hours in BMM with 5 mM CaCl2 or with no added Ca2+. Cells were harvested, washed and resuspended in 10 mM MgSO4, containing the same amount of Ca2+ as the original culture to obtain an OD600 of 0.2. One end of the midrib was inoculated with the resuspended culture and the other end of the midrib was inoculated with sterile 10 mM MgSO4 with 5 mM Ca2+ or no added Ca2+, as controls. The Petri dishes were placed in a clear plastic bin with water to maintain humidity. The bins were incubated at room temperature with ambient sunlight for six days after which the zones of disease were measured. The experiments were performed in at least three independent biological replicates, and the averaged values with standard deviation are presented.The lettuce infection model with the following modifications was used to assess strains pathogenicity efhP lacking mutant PAO1043 were transformed with pMMB66EH (courtesy of Dr. Delfina Dominguez), carrying aequorin PAO1 and its 2+]in was performed as described in 2+]in, the measurements were recorded for 1 min at 5 sec interval, then the cells were challenged with 1 mM Ca2+, mixed for 1 sec, and the luminescence was recorded for 20 min at 5 sec interval. Injection of buffer alone was used as a negative control, and did not cause any significant fluctuations in [Ca2+]in. [Ca2+]in was calculated by using the formula pCa = 0.612(−log10k)+3.745, where k is a rate constant for luminescence decay (s-1) 2. The luminescence released during the discharge was monitored for 10 min at 5 sec interval. The estimated remaining available aequorin was at least 10% of the total aequorin. The experimental conditions reported here were optimized to prevent any significant cell lysis.Luminescence measurements and estimation of free in to nearly basal WT level, as observed in WT PAO1 cells. Instead, the [Ca2+]in continued to raise and in 20 min reaching 3.66±0.41 µM, which is almost seven fold higher than in PAO1. This suggests that EfhP is involved in maintaining intracellular Ca2+ homeostasis, which supports its predicted Ca2+-binding capabilities.The role of EfhP in maintaining intracellular Caaequorin Fig. 2. P. aeruginosa, we performed global quantitative proteomics on wild-type P. aeruginosa strains PAO1 and the CF isolate FRD1. We compared those proteomes to the proteomes of their respective efhP deletion mutants, designated here as PAO1043 and FRD1043, respectively. Since our previous proteomics studies showed an effect of both [Ca2+] and mode of growth (biofilm versus planktonic) on proteome profiles of the wild-type strains 2+] (no added Ca2+) and high [Ca2+] (10 mM), planktonically and in silicon tubing biofilms 2+. 2+, based on our prior study In order to determine the role of EfhP in the physiology of efhP mutant strain showed any significant difference in proteome profiles compared to the respective wild-type strain, when cultivated in medium with no added Ca2+. However, when cultured with 10 mM Ca2+, eight proteins that were abundant in PAO1 were absent in the proteomes of PAO1043 biofilm and/or planktonic cultures: PvdNO, FptA, PA1069, Pfp1, and PhzB1B2D1  that were absent from the efhP mutant. In tubing biofilms, twenty-nine protein differences were observed in the FRD1043 mutant versus wild-type strains . However, the lack of efhP impaired the resistance of FRD1 to H2O2 treatment when cells were cultured in the presence of 10 mM [Ca2+], and FRD1043 showed a 60% decrease in survival when exposed to 1 mM of H2O2 for 60 min . Considering that Ca2+ accumulates in pulmonary liquids of CF patients P. aeruginosa adaptation to a host environments with elevated [Ca2+].The 2+-binding protein spanning the inner membrane, with the two EF-hand domains facing the periplasm. Deletions of efhP in two P. aeruginosa strains causes multiple changes in the cytosolic proteome of both strains, but with more changes occurring in the CF pulmonary isolate FRD1. The effects of the efhP deletions only occurred when the cells were exposed to elevated [Ca2+] and included reduced abundance of virulence factors and stress response proteins. The lack of efhP abolished production of pyocyanin, and reduced the degree of infection, biofilm formation, and resistance to oxidative stress in FRD1 at high [Ca2+]. The mutant also lost the ability to produce alginate at no iron and high [Ca2+]. Finally, the lack of EfhP abolished the ability of P. aeruginosa to maintain intracellular Ca2+ homeostasis. These findings suggest that EfhP is important for Ca2+ homeostasis and plays role in Ca2+- triggered virulence and resistance of P. aeruginosa in high Ca2+ environments.Sequence analyses predicted that EfhP is a CaFigure S1Sequence alignment of EfhP and its homologs from nine Pseudomonas strains. EF-hands are shown in boxes, transmembrane regions are shown in grey. Conserved amino acids are in bold. Sequences were aligned using ClustalW.(TIF)Click here for additional data file.Figure S2Genome context of the efhP homologs from Pseudomonads. The organization of the efhP genomic neighborhood in P. putida strains KT2440, F1, W619, and GB-1, P. entomophila L48, P. aeruginosa strains LESB58, PAO1, and PA7, P. fluoresecens Pf0-1, and P. syringae 1448A. Genes are colored according to their predicted functional domains: black, EfhP and homologs; wavy lines, DUF domain proteins; horizontal lines, transcriptional regulators; diagonals, two component system; light grey, hypothetical proteins.(TIF)Click here for additional data file.Figure S3Biofilm assessment. Biofilm formation was quantified using 96-well plates. Cells were cultured on BMM medium containing either 10 mM CaCl2 or no added calcium for 24 h with medium replacement at 12 h. Biofilms were stained with crystal violet, and the absorbance was measured at 595 nm using a microtiter plate reader.(TIF)Click here for additional data file.


A fall in mitogen-activated protein kinase (MAPK) activity is essential for pronuclear formation, but the precise timing and mechanism of decline are unknown. Here, we have measured the dynamics of MAPK pathway inactivation during fertilization of mouse eggs using novel chemiluminescent MAPK activity reporters. This reveals that the MAPK activity decrease begins during the Ca2+ oscillations, but MAPK does not completely inactivate until after pronuclear formation. The MAPKs present in eggs are Mos, MAP2K1 and MAP2K2  and MAPK3 and MAPK1 . Notably, the MAPK activity decline at fertilization is not explained by upstream destruction of Mos, because a decrease in the signal from a Mos–luciferase reporter is not associated with egg activation. Furthermore, Mos overexpression does not affect the timing of MAPK inactivation or pronuclear formation. However, the late decrease in MAPK could be rapidly reversed by the protein phosphatase inhibitor, okadaic acid. These data suggest that the completion of meiosis in mouse zygotes is driven by an increased phosphatase activity and not by a decline in Mos levels or MEK activity.Egg activation at fertilization in mammals is initiated by prolonged Ca Before fertilization, mammalian oocytes are arrested at the second metaphase of meiosis. Upon fertilization, fusion with the sperm causes the egg to complete meiosis and the zygote to enter the first interphase of embryo development. The unfertilized egg is arrested in metaphase II as a result of the continuous activity of two main types of protein kinase . One of 2+ increase in the egg cytosol . In mammalian eggs, there is substantial evidence suggesting that the sperm causes these Ca2+ oscillations by the introduction of a sperm-specific phospholipase C, PLCζ, after gamete membrane fusion (2) to drive repetitive cycles of inositol trisphosphate (IP3) production and Ca2+ release that last for several hours , which leads to phosphorylation of Emi2 and eventually results in Emi2 proteolysis  compared to that of MAPKAR  in each of the ERK1 monomer sequences was produced . This re stimuli . The subresidues .AR-injected eggs treated with U0126 were next correlated with the dephosphorylation patterns of both the endogenous ERK1 and recombinant MAPKAR. The immunoblots in ARΔ4 was also dephosphorylated at the same time as the endogenous ERK1 in mouse eggs exposed to U0126 , the MAPKAR luminescent probes can be regarded as surrogate reporters for the state of ERK1 phosphorylation and, hence, the degree of activation of MEK, the penultimate kinase in the MAPK pathway.The luminescence changes in MAPKto U0126 . We noteAR and MAPKARΔ4, we then examined the timing of the decrease in MAPK activity during fertilization. We carried out experiments where the luminescence of our MAPK probes was measured alongside the fluorescence of a Ca2+-sensitive dye. 2+ oscillations and the luminescence signal changes from MAPKAR. Images of the luminescent eggs before and after fertilization are shown in the insets in 2+ transients. After a couple of hours, there was a change in luminescence that started before, but was not completed until well after, the cessation of Ca2+ oscillations. In fertilized eggs where we monitored the full series of Ca2+ oscillations and change in MAPKAR signal, the time period from the first Ca2+ increase to the cessation of Ca2+ oscillations was 4 h 5 min , whereas the time that had elapsed between the first Ca2+ transient and the half maximal change in MAPKAR luminescence was 6 h 38 min . The change in luminescence commenced at between 2 and 3 h after the initiation of Ca2+ oscillations. In every experiment where there was a detectable luminescence signal at the beginning of Ca2+ oscillations, the inflection point indicating the start of the change in MAPKAR luminescence always occurred before the last of the series of Ca2+ oscillations. In many, but not all cases, there appeared to be two phases of luminescence change with one phase before and another after the cessation of Ca2+ oscillations.Using MAPKARΔ4 . Owing to the higher initial luminescence values, we were able to estimate that the decline in MAPKARΔ4 signal started 2 h 25 min  after the first Ca2+ increase at fertilization. This time point corresponds to the occurrence of the 18th Ca2+ transient out of 25 Ca2+ transients  suggesting that the change in MAPK begins about three quarters of the way through the total number of Ca2+ transients. The fertilization-induced luminescence signal with MAPKARΔ4 also showed signs of a two-phase change, with one phase before and another after the cessation of Ca2+ oscillations. It is known that the cessation of Ca2+ oscillations in mouse zygotes correlates with the time of pronuclear formation  These data show that the changes in luminescence we see with the MAPKAR and MAPKARΔ4 probes reflect phosphorylation of ERK1. Consequently, our data with two different ERK1-based reporters suggest that the onset of MAPK inactivation starts abruptly towards the end of the series Ca2+ oscillations at fertilization, a point well before pronuclear formation. However, the MAPK inactivation then continues progressively for a considerable time after pronuclear formation.Fertilization experiments were also conducted using MAPKARΔ4 . With thllations . The statch e.g. . Eggs inormation . It was 2+ oscillations measured with a fluorescent dye. Because Mos is considered to be present at a very low concentration in eggs, we injected small amounts of Mos–luc cRNA to produce a low luminescence signal consistent with the level of reporter expression  fusion protein, in a similar approach to previous studies of cyclin B stability . This is not significantly different to the 2 h 2 min  observed for the corresponding time for control eggs not expressing Mos–Cherry, and which were simultaneously fertilized in the same experiment. The average level of Mos–Cherry fluorescence in all these experiments was ∼5 times greater than the egg autofluorescence. These data suggest that Mos expression in mouse eggs does not affect the onset or the subsequent time course of the decline in the MAPK pathway activity at fertilization.We next wanted to investigate whether overexpression of Mos would perturb the temporal profile of the MAPK decrease or the formation of pronuclei. Mos overexpression has previously been shown to block MAPK inactivation in fertilizing ascidian eggs, and this response is seen with Mos from a variety of species . We were oocytes . Fig. 5A2+ transients initiate the events of metaphase–anaphase transition, and the termination of Ca2+ oscillations is precisely correlated with pronuclei formation in mouse eggs, we again used the start and cessation of Ca2+ oscillations as a proxy for measuring the period required for eggs to form pronuclei and enter interphase. 2+ oscillations recorded from a fertilizing mouse egg injected with a high concentration of Mos–luc cRNA to induce luminescence expression levels ∼1000 times higher than in the above experiments  with 46.5  Ca2+ spikes for eggs with high levels of Mos–luc injected , compared to 3 h 18 min  and 46.9  Ca2+ spikes for 18 parallel control eggs. Consequently, these results indicate that overexpression of Mos–luc does not alter the timing of pronuclei formation. Combined with the above measurements of Mos–luc decline, our data suggest that increased Mos destruction does not occur in response to sperm-induced Ca2+ signals. In addition, the continued presence of Mos protein in eggs does not affect the timing of MAPK activity decrease, the occurrence and termination of Ca2+ oscillations, or the formation of pronuclei.We also examined whether the mouse Mos–luc itself could affect the time course of pronuclear formation because this depends upon a decline in MAPK levels . Becauseeriments . The Ca2oduction . These M2+ oscillations, we next determined whether the dynamics of MAPK decline was influenced by the presence or absence of Ca2+ signals. When Ca2+ oscillations are initiated in fertilized eggs, they can be stopped by addition of sufficient BAPTA to chelate extracellular Ca2+  for BAPTA-treated eggs (n = 33) which is slightly longer than the mean time for control eggs reported above . This data suggests that whereas premature termination of Ca2+ oscillations still allows MAPK inactivation, the kinetics of inactivation are slightly delayed by the absence of Ca2+ signals.Because the MAPK decline starts during the Calar Ca2+ , but not2+ oscillations early can be achieved by inhibiting pronuclear formation. In wheat germ agglutinin (WGA)-injected mouse eggs, pronuclear formation is prevented and Ca2+ oscillations persist for a prolonged period owing to the absence of nuclear localization of PLCζ  for WGA-injected eggs (n = 29), which is not significantly shorter than control fertilizing eggs. This result suggests that the ongoing presence of Ca2+ oscillations has rather little influence upon the start of MAPK inactivation.The converse situation to stopping the fertilization-induced Ca of PLCζ . Fig. 6B2+ increase . The delay from the addition of roscovitine to the start, and to the half maximal change in signal, in three eggs was ∼6 h for the MAPKAR signal but was hard to define due to the lack of initial basal luminescence with MAPKAR. The delay to the start of the MAPKARΔ4 signal was 3 h 35 min  after addition of roscovitine. The response to roscovitine was more variable than at fertilization, but within the same range of times. These data, therefore, suggest that the change in MAPKAR and MAPKARΔ4 signals after roscovitine addition was similar to that seen after fertilization, and is clearly much more delayed compared with the addition of U0126 . In ARΔ4 signal had started to change, we found that there was change in luminescence, which reverted back to unfertilized levels. There was also a persistence of Ca2+ oscillations after adding okadaic acid, which is probably caused by the fact that okadaic acid prevents pronuclear formation, which blocks PLCζ nuclear localization . These data suggest that okadaic acid can prevent a decrease in ERK1 activity after the initiation of the decline in ERK1 signaling. Because ERK1 phosphorylation is restored after okadaic acid application, this implies that a kinase activity is still able to phosphorylate ERK1 even when it is normally in the process of being dephosphorylated. MEK is clearly the crucial kinase required for restoring ERK1 activity as shown by experiments on unfertilized eggs. As shown previously  and luminescence . Consequently, our probes cannot be interpreted as directly monitoring ‘dimerization’ of ERK1 subunits given that one assumes this should involve subunits coming together and lead to an increase in luminescence. The importance of phosphorylation for the change in luminescence is shown by the observation that a mutant MAPKAR, which lacks the T202 and Y204 phosphorylation sites . These data show that ERK1 phosphorylation and activation are reported effectively by our MAPKAR proteins.There are a number of probes that consist of a single molecule with the N- and C-termini of luciferase at each end, and a pair of nested proteins that bind to each other . In someon sites , did notARΔ4 reporter showed a higher basal luminescence level than the wild-type MAPKAR reporter. This suggests that phosphorylation can still trigger a conformation change between the two ERK1 protomers. We can assume that there is likely to be a greater degree of complementation between the split domains of luciferase with the MAPKARΔ4 reporter. Notably, it has been previously shown that the Δ4-ERK1 protein can be dephosphorylated at the same rate as wild-type ERK1  and the formation of pronuclei, which occurs ∼3 h later . All PCR-amplified ERK1 constructs were first inserted into the pCR®-Blunt II-TOPO® vector (Invitrogen) and then subcloned into the pCR3 vector (Invitrogen). The denoted restriction sites were used in construction of the ERK1 luciferase construct: BamHI–N-Luc(1–413)-EcoRI–ERK1–EcoRI–16GS–ERK1–XhoI-C-Luc(395–543)–XbaI. The construct is referred to as the MAPK activity reporter (MAPKAR). A control ERK1–luciferase construct that is deficient in its ability to dimerize was prepared using the dimerization-deficient mutant ERK1Δ4(Pro193–Asp196) template which was amplified from the GFP–ERK1-Δ4 plasmid kindly provided by Phillipe Lenormand  was generated with a Quick-Change Mutagenesis kit (Stratagene) using the oligonucleotide primers 5′-GGCTTCCTGGCGGAGTTTGTGGCTACGCG-3′ and 5′-CCCTCGCCGATGTACTGCAACTGCGTGTA-3′. This is referred to as MAPK-DM. Plasmids encoding the mouse Mos (In vitro synthesis of capped RNAs was performed using Nde1-linearized plasmids with the T7 mScript™ Standard mRNA Production System (Cambio) as per the manufacturer's instructions, and these RNAs were then polyadenylated as described previously  . This consisted of a stock of concentration of 1 mM in KCl/Hepes buffer that was then diluted 1∶1 with cRNA for a particular luciferase construct. The luminescence and fluorescence measurements were recorded from eggs using one of two intensified charge-coupled device (ICCD) photon-counting imaging systems  that switch between recording modes and have been described previously (2+ oscillations and not their absolute magnitude. For in vitro fertilization experiments, data were only analyzed for eggs where the Ca2+ oscillations could be recorded from start to finish without a major deviation of the baseline, which otherwise occurred occasionally in eggs that moved during recording. Data was collected and analyzed using the Photek camera software and SigmaPlot. Further details of the imaging system and methodology has been given previously (2+ oscillations seen in control fertilizations, and so key experiments compared interventions with groups of eggs studied within the same month as control eggs.In our experiments, the luminescence of eggs depicts the emission from the recombinant luciferase-tagged proteins expressed from the cRNAs that we injected. Eggs were incubated in M2 medium for 2–3 h to allow expression of the luciferase-based constructs before starting an experiment. The fluorescence recording is to detect the Caeviously . Mos–Cheeviously . Unless eviously . These e


Calaxin is an opisthokont-specific neuronal calcium sensor protein first described in the sperm of the ascidian Ciona intestinalis. It binds to a heavy chain of two-headed outer arm dynein in a Ca2+-dependent manner and regulates ‘asymmetric’ wave propagation at high concentrations of Ca2+. A Ca2+-binding subunit of outer arm dynein in Chlamydomonas reinhardtii, the light chain 4 (LC4), which is a Ca2+-sensor phylogenetically different from calaxin, shows Ca2+-dependent binding to a heavy chain of three-headed outer arm dynein. However, LC4 appears to participate in ‘symmetric’ wave propagation at high concentrations of Ca2+. LC4-type dynein light chain is present in bikonts, except for some subclasses of the Excavata. Thus, flagellar asymmetry-symmetry conversion in response to Ca2+ concentration represents a ‘mirror image’ relationship between Ciona and Chlamydomonas. Phylogenetic analyses indicate the duplication, divergence, and loss of heavy chain and Ca2+-sensors of outer arm dynein among excavate species. These features imply a divergence point with respect to Ca2+-dependent regulation of outer arm dynein in cilia and flagella during the evolution of eukaryotic supergroups.The motility of eukaryotic cilia and flagella is modulated in response to several extracellular stimuli. Ca Cilia and flagella are eukaryotic machineries for cell motility propelled by the propagation of bending waves. The internal cytoskeletal structures, called axonemes, are constructed from 9+2 microtubules with axonemal dyneins and regulatory structures such as the central apparatus and radial spokes . These s2+ is an important factor for these modulations. Here, I first introduce the diversified roles of Ca2+ in ciliary and flagellar motility over several eukaryotes and then focus on the Ca2+ sensors that directly regulate the motile machinery, the axonemes. In addition, I present a phylogenetic analysis of Ca2+ sensors, demonstrating the evolution of Ca2+ sensors and proposing a pathway of eukaryotic evolution.Motility of cilia and flagella is modulated by several extracellular stimuli to enable directed and harmonious movement of cells and tissues. Ca2+ is a well-known intracellular regulator for modulation of ciliary and flagellar movements. These modulations range across diverse modes, including (1) changes in ciliary or flagellar waveforms, (2) rotation or reversal of the direction of ciliary or flagellar bending, (3) arrest of beating, and (4) increasing of beat frequency  ahead of the direction of movement for fertilization of the egg. Sperm of the ascidian  the egg ,10. ThisChlamydomonas reinhardtii has two flagella and usually swims in breast stroke fashion with the flagella located anterior to the cell body. A structure for mating is formed between the two flagella at fertilization [Chlamydomonas stops its motility and then moves in the reverse direction with conversion of flagella into a symmetrical waveform [2+ concentration. The increase of intracellular Ca2+ appears to be performed by Ca2+ influx through a voltage-dependent channel CAV2 [2+ is observed in the prasinophyte Spermatozopsis similis [The unicellular alga lization ,12. Whenwaveform . Analysinel CAV2 . Similar similis .Paramecium, Ca2+ causes reversal of the beating plane of cilia [2+ influx, resulting in ciliary reversal and backward swimming. It is considered that ciliary reversal in Paramecium cilia is caused by the rotation of the central pair in the axoneme [2+-dependent manner [In of cilia -18. Extr axoneme ,20. Rotat manner . Howevert manner .Trypanosoma propagate flagellar waves both from the base to tip and the tip to base [2+ in the trypanosomatid Crithidia oncopelti [ to base ,24. Demencopelti .2+-dependent manner [Strombus luhuanus, the reversal of bend propagation appears to be involved in sperm release from the sperm storage site in the female genital tract [Sperm in some insects and snails reverse the direction of bend propagation in a Cat manner -30. For 2+. Spontaneous arrest of mussel gill cilia is caused by membrane depolarization, depending on calcium ions [−4 M Ca2+ [Ciona stigmatal cells also depends on the presence of external Ca2+ [2+ ionophore A23187 in seawater [Epithelial cilia of marine invertebrates show ciliary arrest in response to Caium ions ,32. Most4 M Ca2+ . Ciliarynal Ca2+ . Cilia oseawater .2+ induces increased beat frequency in airway cilia in mammals [Paramecium model and is inhibited by a calmodulin (CaM) antagonist [2+ in sea urchin [Ciona , although a demembranated model of sea urchin sperm flagella changed to an asymmetric waveform on stimulation with Ca2+, and showed quiescence at Ca2+ concentration >10−4 M [Ca mammals -38 and i mammals , withouttagonist . However >10−4 M .2+ on ciliary and flagellar motility appear diverse among organisms, but the roles of Ca2+ in the regulation can be classified into two parts. One is a signaling pathway upstream of the modulation of the axonemes. Influx of Ca2+ is an important trigger for the modulation of ciliary and flagellar motility. Several Ca2+ channels and Ca2+-binding enzymes, such as protein kinases and phosphatases, have been reported to be localized and functional in the ciliary/flagellar plasma membrane and the ciliary/flagellar matrix [2+-binding proteins such as calaxin, dynein light chain 4 (LC4), CaM, and centrin are bound to the substructures of the axonemes and directly modulate dyneins or their regulatory elements, the radial spokes, and central apparatus. In this paper, I focus on the Ca2+ sensors that directly act on the outer arm dynein in the axonemes.The effects of Car matrix ,7. The oThe extent of flagellar or ciliary bending correlates with the velocity of microtubule sliding ,42. The Chlamydomonas CP mutants [Chlamydomonas flagella have shown that signals from the central apparatus activate specific dyneins for local bending [The central apparatus (CP) - along with the radial spokes (RS) - plays an important role in flagellar motility as revealed by the paralysis of  mutants ,45. The  mutants ,47, and  mutants . The act mutants -51. Stud bending . As prev bending ,54.Chlamydomonas and in the sperm of Ciona and sea urchins [Chlamydomonas, respectively. Other subunits, including intermediate chains and light chains, are involved in the assembly and regulation of dyneins. Several studies with Chlamydomonas mutants and outer arm extracted sea urchin sperm indicate that outer and inner arm dyneins are involved in the elevation of microtubule sliding velocity (increasing beat frequency) and formation and propagation of flagellar bending, respectively [Axonemes have two dynein motors with different properties: outer arm dynein and inner arm dynein. Subunits of the outer arm dynein have been well studied in  urchins ,55-59. Tectively ,4.2+-dependent modulation of ciliary motility. The conversion of flagellar wavelength from symmetric to asymmetric is transiently observed during chemotaxis of the sperm to the egg [2+-dependent regulation of outer arm dynein (see below). Lack of outer arm dynein in the human sperm causes low swimming velocity, loss of circular movement with an asymmetric waveform, and low efficiency of penetration into the egg coat [Much experimental evidence demonstrates that outer arm dynein is essential for Ca the egg ,60,61. Tegg coat ,63.Chlamydomonas changes swimming direction in response to light. There are two types of response: a photophobic reaction to very strong light, photoshock, and a positively or negatively directed movement towards a light source, phototaxis. Both photoshock and phototaxis depend on changes in intracellular Ca2+. Reactivated Chlamydomonas axonemes show an asymmetric beat pattern at Ca2+ concentrations below 10−6 M, become quiescent at 10−5 M, and then resume beating with a symmetric waveform at 10−4 M [Chlamydomonas cell models differentially respond to Ca2+ concentration in the range 10−8 M to 10−6 M [t 10−4 M . This wat 10−4 M ,59,65. Io 10−6 M . Studieso 10−6 M ,59.Trypanosoma brucei results in the loss of tip to base propulsive propagation of the flagellar wave [2+-dependent, and the base to tip propagation is only observed in demembranated models when demembranated and reactivated in the presence of EGTA [Schmidtea mediterranea demonstrated that outer arm dynein is essential for the increase of beat frequency and coordination of cilia to produce ciliary oscillation with metachronal waves [Specific knockdown of outer arm dynein LC1 in lar wave  that is lar wave . The tip of EGTA . RNAi knal waves .2+ are mediated by Ca2+-binding proteins. The most common motif for Ca2+ binding is the EF hand. It is a helix-loop-helix structural motif of 12 residues (+X)x(+Y)x(+Z)x(−Y)x(−X)xx(−Z) for metal coordination, where +X, +Y, +Z and −X, −Y, −Z are the vertices of an octahedron [2+ in CRP and does not match the consensus sequence in a PROSITE search  in Chlamydomonas, the outer dynein could not be isolated from sperm flagella in association with any Ca2+-binding proteins. Moreover, conversion into an asymmetric flagellar waveform is achieved at high concentrations of Ca2+ in the sea urchin sperm model demembranated by Triton X-100 in the presence of millimolar Ca2+ [2+ but become asymmetric when Ca2+ is increased in the reactivation medium. The asymmetric flagellar waveform is seen only in the presence of high concentrations of ATP [Many studies showed that CaM is an important Caovements ,75. Althlar Ca2+ . In thiss of ATP , which i2+-dependent conversion of flagellar waveform is essential for sperm chemotaxis [Ciona intestinalis, correlation between the increase in intracellular Ca2+ concentration and conversion of flagellar asymmetry is clearly observed [Ciona sperm show rather planar wave propagation in seawater with a slight asymmetric flagellar waveform, resulting in a circular trajectory. The reception of the gradient of chemoattractant  from the egg [2+ concentration. Flagellar axonemes respond to the change and temporarily form and propagate an asymmetric waveform, resulting in a turning movement towards the egg [Caemotaxis ,60,76-79emotaxis , responsemotaxis , self-noemotaxis , hyperacemotaxis ,84, and emotaxis ,86. In t the egg  induced 2+-binding protein that is expressed in Ciona testis during the course of extensive descriptions of axonemal proteins [2+-binding axonemal protein calaxin [A previous study found a Caproteins . It turn calaxin . Calaxin calaxin ,91. A ph2+-binding EF hand motifs  [2+-binding to these sites was directly demonstrated by isothermal titration calorimetry (ITC), showing a three-site sequential binding model [2+-dependent hydrophobic interactions are suggested from positive enthalpy in ITC, as in the case of Ca2+ binding to calmodulin [2+, allowing them to associate with membranes. The consensus sequence for myristoylation, N-terminal GXXXSX [2+-myristoyl switch property of NCS. Immunohistochemical observations indicate that calaxin is located on the outer arm dyneins along the axoneme of sperm flagella [2+ and to the β heavy chain (ortholog of the Chlamydomonas γ heavy chain) of the outer arm dynein [a. Calaxin binds to the N-terminal stem region, as revealed by far-western blotting against UV-cleaved fragments of the β heavy chain . Although two IQ consensus motifs for binding CaM-like proteins are located within the stem domain of the Chlamydomonas γ heavy chain [Ciona β heavy chain.Calaxin has three Cacalaxin) ,89. Ca2+ng model . Two of lmodulin . Severallmodulin ,94. In fflagella . Far wesvy chain , there iCiona sperm shows a unique turning movement associated with a flagellar change to asymmetric waveforms, followed by straight-ahead movement towards the chemoattractant SAAF [−6 M Ca2+. Repaglinide attenuates propagation of asymmetric waveforms, but not the relatively symmetric waveforms seen at low concentrations of Ca2+. Calaxin directly suppresses the velocity of microtubule sliding by outer arm dynein at high Ca2+ concentrations. Repaglinide and anti-calaxin antibody cancel the suppression of microtubule translocation at high concentrations of Ca2+. All of these data demonstrate that calaxin plays an essential role in the propagation of asymmetric flagellar bending by suppression of dynein-driven microtubule sliding at high concentration of Ca2+ [ant SAAF . In the ant SAAF . Repagli of Ca2+ . Calaxin of Ca2+ . Then, hCiona calaxin in the genomes of Chlamydomonas reinhardtii and Paramecium tetraurelia hit hypothetical proteins CHLREDRAFT_119565 (XP_001696107) (E = 4e−13) and XP_001433234 (E = 2e−15), respectively. Both hypothetical proteins show a best match with calcineurin subunit B type 1-like protein (CBL-1), not calaxin, in the Ciona genome. LC4 is a Ca2+-binding subunit of outer arm dynein first identified in Chlamydomonas [2+-binding assays demonstrate that LC4 has at least one functional Ca2+-binding site. LC4 is isolated in association with the γ heavy chain of outer arm dynein. These properties suggest functions of LC4 analogous to those of calaxin, although the proteins are phylogenetically distinct from each other.BLASTP searches for 5 XP_0016107 (E = Chlamydomonas) or Stramenopiles . Calaxin homologs were only found, and were well conserved, in species of the opisthokont supergroup, including Homo sapiens, Mus musculus, Ciona intestinalis, Strongylocentrotus purpuratus, Amphimedon queenslandica, Drosophila melanogaster, Monosiga brevicollis, and Crassostrea gigas. The opisthokont organisms that lack motile cilia or flagella throughout their life cycles, such as C. elegans, Vericrustaceans (except Notostraca and Thecostraca), yeast, and higher fungi show no calaxin gene in their genomes, although these organisms have genes for other NCSs such as NCS-1 (frequenin). The chytrid fungus Batrachochytrium dendrobatidis, grouped into the opisthokonta with metazoa, contains a calaxin gene (XP_006677085) in its genome. The calaxin of B. dendrobatidis shares 38% amino acid identity with Ciona calaxin  and bikonts , based on the concept of eukaryotic cells with a single flagellum or two flagella, respectively. Opisthokonts are groups shown to propel cells by a posterior flagellum -99. Homom dynein . Howeverm dynein -103, it A previous study identified proteins with a unique combination of domains: an intermediate chain of outer arm dynein, thioredoxin domain and nucleoside diphosphate kinase domain  andChlamydomonas. In the present study, an attempt has been made to biochemically compare the outer arm dynein and its Ca2+ sensor between Ciona sperm flagella and Chlamydomonas flagella and to correlate their functions in the regulation of motility.Knowledge of the molecular components of axonemal dyneins and the molecular mechanism of ciliary and flagellar motility has been accumulated mostly from metazoan sperm and certain protists such as Ciona sperm flagella consists of two heavy chains and represents a two-headed structure, but that of Chlamydomonas flagella consists of three heavy chains with a three-headed structure. Each of the two heavy chains of sperm outer arm dynein is known to have distinct properties [Ciona β and Chlamydomonas γ heavy chains) mediates structural and rigor binding to the microtubules [In vitro motility assays indicate that the absence of the Chlamydomonas γ heavy chain increases both microtubule gliding and ATPase activity [The outer arm dynein of operties -110. Seaactivity , suggestCiona calaxin and Chlamydomonas LC4 bind to Ciona β and Chlamydomonas γ heavy chains, respectively [2+ dependency of the binding is reversed between Ciona and Chlamydomonas  in the presence of Ca2+ [2+ binding to LC4 on dynein-driven microtubule sliding has not been examined in Chlamydomonas, the binding of Ca2+ to LC4 induces activation of ATPase activity of the outer arm dynein in the mutant lacking the α heavy chain [2+-dependent regulation of the γ heavy chain; in the absence of Ca2+, LC4 is tightly bound to the γ HC, resulting in inefficient formation of a rigor bond with microtubules. In the presence of high Ca2+, Ca2+-bound LC4 detaches from the IQ region of the γ heavy chain and becomes attached to IC1, resulting in a structural change of the N-terminal stem domain and the activation of motor activity [ectively ,112. How of Ca2+ . The bin of Ca2+ . In the  of Ca2+ ,112. Altactivity .Figure 42+ on the regulation of outer arm dynein in Ciona and Chlamydomonas are likely to connect with the difference in the changes of flagellar waveforms . For example, sperm of freshwater fish show asymmetric waveforms depending on an increase in intracellular Ca2+ concentration [Pyramimonas parkae shows waveform conversion similar to Chlamydomonas reinhardtii [2+ concentration has not been elucidated. An interesting experiment was the examination of the relationship between intracellular Ca2+ concentration and flagellar waveform in the prasinophyte algae Pterosperma and Cymbomonas, both of which show conversion of flagellar waveforms similar to metazoan sperm: symmetric flagellar waveforms in normal swimming and asymmetric waveforms when they change swimming direction [2+ is unclear.It is unlikely that ciliary response in waveform conversion depends on the extracellular Cantration ,114. Thenhardtii , althougirection . Anterioirection . These oirection . They chirection , but theCiona calaxin, CaM, centrin, NCS, calcineurin B-subunit (CN-B), Chlamydomonas LC4, and a Ca2+-binding subunit of outer arm dynein docking complex 3 (DC3) [Chlamydomonas LC4 and its orthologs were grouped into a clade different from that of calaxin but were more closely related to calaxin than were CaM or centrin. BLASTP searches of Chlamydomonas LC4 against genomes of bikonts resulted in finding orthologs in flagellated species including ciliates, dinoflagellates, diatoms, brown algae, haptophytes, and cryptophytes. Exceptions are seen in organisms lacking outer arm dynein such as angiosperm, moss, and fern [Chlamydomonas LC4 against these species resulted in best hits to CaM. Search of Chlamydomonas LC4 in the genomes of opisthokonts failed to find any homologs in this supergroup. For example, the protein most homologous to LC4 in Ciona intestinalis was CaM (E = 3e−22).A phylogenetic analysis of  3 (DC3) ,120 usinand fern . BLASTP 2+-binding with a ratio of 1 mol Ca2+/mol protein [2+ under physiological conditions because it also significantly binds Mg2+ [Plasmodium) and Cryptophytes but could not be found in the Ciona or human genomes. DC3 grouped into a clade closer than LC4 to CNB/calaxin/NCS , although ultrastructural observation of the flagella clearly shows the presence of outer arm dynein [Plasmodium (Apicomplexa).DC3 is also a CaM type of EF hand protein localized on the outer arm dynein docking complex and shows redox-sensitive Ca protein . Howevernds Mg2+ . Genes oS Figure . Intrigum dynein . LC4 was2+ signaling [Chlamydomonas (ID391130) and in Paramecium (GSPATP9660001) are grouped with CNB-like protein. Separation of these proteins from the CNB group is supported by the bootstrap value .Both CN-B and NCS have been found in animals and fungi  but do nignaling . CNB-likEuglena, Trypanosoma), Heterolobosea , and Tsukubamonadida. The Metamonada includes amitochondriate flagellate Fornicata , Parabasalids , and Preaxostyla [The supergroup Excavata includes eight taxa -128. Phyaxostyla . Althoug2+ sensors in Excavata leads to an interesting point of view concerning the evolution of Ca2+ sensor proteins  and Naegleria gruberi (ID 70962) contain clear orthologs of Chlamydomonas LC4 . The ouflagella ,138, indflagella ,131 indii et al. , the numPhysarum possess 9+2-structured flagella. Cross sections of Physarum axonemes suggest that the outer arm dynein is two-headed [Chlamydomonas LC4 or DC3 in the chlorarachnion Bigelowiella natans. The number of heavy chains is possibly three judged from an EM image [Plasmodiophora brassicae, apparently possesses three-headed outer arm dynein [Paramecium and Tetrahymena, have three-headed outer arm dynein and a gene orthologous to Chlamydomonas LC4. However, another group of Alveolata, the Apicomplexa, shows a different feature; the axonemes of Plasmodium berghei have normal 9+2 structure with three-headed outer arm dynein [P. berghei has LC4 since the genome sequence of this organism is not available. The gregarin Lecudina tuzetae has a 6+0 structured axoneme, but the detailed structure of the outer arm dynein is unclear from the available EM images [It is believed that the two heavy chains of the ODAβ family resulted from gene duplication , but theo-headed -103, likEM image . Anotherm dynein , but no m dynein . It is nM images .Euglena, Leishmania, and Trypanosoma show a two-headed shape of outer arm dynein. The genome sequences reveal that neither Euglena nor Trypanosoma have LC4. Second, Giardia has an LC4 homolog in the genome. EM images, however, are very close to those of two-headed outer arm dynein [Giardia lamblia is a fast-evolving parasitic species, leading to an error in phylogenetic analysis due to long-branch attraction (LBA) [Naegleria gruberi and Trichomonas vaginalis, appear three-headed, although little TEM data with clear images of outer arm dynein is available [Six species in the Excavata were available for prediction of the number of heavy chains from EM images. First, the euglenozoan species m dynein ,144. Thion (LBA) . Lastly,vailable ,147.2+; examples of such regulated movement are reversal of bend propagation and altering of flagellar waveforms  Excavata) have three-headed outer arm dynein with Chlamydomonas LC4-type Ca2+ sensors. Excavata robustly emerge between unikonts and Archaeplastida/Hacrobia/Stramenopiles/Alveolata/Rhizaria and form a monophyletic supergroup [Taking into account the fact that cilia have been inherited through the major pathways of eukaryotic evolution, I here propose a hypothesis for eukaryotic evolution based on phylogenetic analyses of Capergroup . Severalpergroup -158, butpergroup .2+ sensors and shed light on the origin of Ca2+-dependent regulation of cilia and flagella. A phylogenetic analysis in this study showed that excavates had already evolved several Ca2+ sensors, including those with similarities to extant Ca2+ sensors. Based on the widely accepted relationship among excavate species [2+ sensors  during evolution. Loss of dynein heavy chains or Ca2+ sensors in Excavata, possibly by reduction of genomes in obligate parasites [The Excavata is certainly a supergroup that could provide key clues to understanding the evolution of dynein and its Ca species ,158, a ps Figure . This moarasites ,163, is Trichomonas and Giardia). Similarly, CNB/NCS-like Ca2+ sensor homologs must have existed in the last common eukaryotic ancestor. Another route for Discoba diversification might have involved retentions of CNB/NCS-like Ca2+ sensors.The duplication of dynein heavy chains would have occurred at the root of the bikont lineage Figure . From thTrypanosoma brucei and Euglena gracilis are biflagellate but one of the two flagella is highly reduced. There are two flagella in Naegleria gruberi, five flagella in Trichomonas vaginalis, and eight flagella in Giardia lamblia  in orga2+ sensors seems particularly important in cilia and flagella because they participate in gamete motility, essential for the success of reproduction in most organisms. For Ca2+ sensors of outer arm dynein, the functions of calaxin and Chlamydomonas LC4 regulate the motor activity in flagella, but their response to Ca2+ concentration is different. The distribution of these Ca2+ sensors in extant species in eukaryotes is described in the present paper. Calaxin and LC4 appear to be preserved in Opisthokonta and the majority of bikonts , respectively.New genes with novel functions are evolved by gene duplication . Several2+ sensors, except Ciona calaxin and Chlamydomonas LC4. To learn whether evolution of proteins by gene duplication and divergence accompanies or precedes innovation of protein function, it would be fascinating to examine the interaction of an ancient calaxin with microtubules or dyneins.It is possible that these proteins became preserved after protein evolution by gene duplication and divergence because of their specific functions in the interaction with the cytoskeleton and the regulation of a molecular motor. The module-dominant conservation, as seen in axonemes , is poss2+ requires outer arm dynein in Chlamydomonas flagella. Conversion to an asymmetric waveform in sperm flagella is also performed by outer arm dynein at high Ca2+ concentration. Thus, the functions of outer arm dynein are regulated by Ca2+ sensors at high concentrations of Ca2+ in both Chlamydomonas and sperm flagella. Recruitment of Ca2+ sensors to outer arm dynein might have made it possible for the organisms to respond to ‘high’ Ca2+ to modulate flagellar waveforms to change their direction of movement, although the directions of conversion of waveforms are a mirror-image of each other in Chlamydomonas and sperm.Conversion from asymmetric to symmetric movement at high concentrations of Ca2+-sensors might have occurred at an early stage in eukaryotic evolution. The clear distinction in dynein structure and Ca2+ sensors between opisthokonts and bikonts, and their heterogeneity in Excavata, suggests an important role of ciliary regulation in eukaryotic evolution. It is unclear, however, if Ca2+ sensors in Excavata really function in the regulation of outer arm dynein. Outer arm dynein in Trypanosoma is essential for tip-to-base movement, which is induced by ‘low’ intracellular Ca2+. Loss of outer arm dynein results in a defect of tip-to-base movement in response to low intracellular Ca2+. This feature of Ca2+ regulation of outer arm dynein is distinct from that observed in Chlamydomonas and Ciona. Trypanosoma and Naegleria have CaM in flagella called flagellar CaM or CaM-1. CaM is localized in paraflagellar rods and regulates their assembly in Trypanosoma [2+-binding proteins in the regulation of the outer arm dynein in Excavata.In this paper, it is suggested that the duplication and divergence of Capanosoma . HoweverChlamydomonas, the mating structure, sits between two flagella. Both Chlamydomonas and sperm move forward with these mating structures at the leading edge (Figure Chlamydomonas [Chlamydomonas. Although it is not known whether this difference in cell polarity is connected to the mirror-image of Ca2+-dependent regulation between these cells, experiments such as knockout of the calaxin gene in metazoa might give important insights into the evolutionary relationship between cilia and organisms.Calaxin was acquired in Opisthokonta and may participate not only in the regulation of fluid flow mediated by cilia and flagella but also in other phenomena that characterize opisthokonts, such as cell polarity, differentiation of nerve cells, and establishment of body plan. The first definition of Opisthokonta by Cavalier-Smith , that ise Figure . Differeydomonas , implyin2+ sensors for outer arm dynein during evolution, but connections of the phylogenetic view of outer arm dyneins to the function or motility of cilia and flagella have not been completely clarified. Most of the discussion in this paper is based on the assumption that orthologous proteins conserve their function across species, but this is not always the case. For solving such problems, it is evidently necessary to confirm whether the proteins used in phylogenetic analyses in the present study are localized and bound to the ciliary or flagellar axonemes and function as Ca2+ sensors of outer arm dynein. The precise number of heads must also be determined by observation with cryo-electron tomography.The present study implies early events in the diversification of CaaThe nomenclatures of dynein heavy chains are complicated because they were originally named according to the electrophoretic mobility on an SDS-gel (see Table


These findings identified crucial roles for Pak1 in cardiomyocyte Ca2+ handling and demonstrated that it functions through unique mechanisms involving regulation of the post-transcriptional activity of key Ca2+-handling proteins, including the expression of Ca2+-ATPase SERCA2a, along with the speculative possibility of an involvement in the maintenance of transverse (T)-tubular structure. They highlight important regulatory functions of Pak1 in Ca2+ homeostasis in cardiac cells, and identify novel potential therapeutic strategies directed at manipulation of Pak1 signaling for the management of cardiac disease, particularly heart failure.A series of recent studies report novel roles for Pak1, a key member of the highly conserved family of serine-threonine protein kinases regulated by Ras-related small G-proteins, Cdc42/Rac1, in cardiac physiology and cardioprotection. Previous studies had identified Pak1 in the regulation of hypertrophic remodeling that could potentially lead to heart failure. This article provides a review of more recent findings on the roles of Pak1 in cardiac Ca Of these, the p21-activated kinases (Paks) form a group of serine/threonine protein kinases activated by Cdc42 and Rac1, and were first discovered in rat brain tissue two decades ago  and delayed rectifier K+ channel currents to β-adrenergic stimulation by isoproterenol were depressed in comparison to SAN cells infected with control virus, Ad-LacZ. Similarly chronotropic responses to isoproterenol stimulation, reflected in repetitive action potential frequency were depressed in both intact SAN tissue and isolated SAN cells expressing active Pak1, when compared to controls expressing Ad-LacZ  pacemaker cells . Immunoprecipitation studies indicated that Pak1 and PP2A form a complex, leading to the hypothesis that Pak1 may regulate LTCC activity through PP2A action  cascade, inhibitor-1 is a downstream PKA target whose activation results in an attenuation of protein phosphatase 1 (PP1) activity   Ca2+ content and decreased SERCA function, particularly during β-adrenergic stress  current (INCX) following INCX induction by 10 mM caffeine were significantly greater in ckoPak1 than Pak1f/f myocytes under chronic β-adrenergic stress. SR Ca2+content, measured by integration of the INCX records, was reduced in Pak1cko compared to Pak1f/f myocytes in both the absence and presence of chronic β-adrenergic stress. The decay rate constants of systolic Ca2+ transients in Pak1cko myocytes kSERCA, representing the kinetics of cytosolic Ca2+ removal brought about by both SERCA and NCX, was significantly lower than that shown by Pak1f/f myocytes in both the absence and presence of chronic β-adrenergic stress ; changes which were not observed in Pak1−/− atrial myocytes. Such findings are consistent with the central role of T-tubules in triggering and synchronizing excitation–contraction coupling, and merit further exploration. T-tubular remodeling has been reported in other cardiac pathologies, and could well be involved in the associated alterations in cellular processes, and hence cardiac function.Preliminary evidence (DeSantiago et al., 2+ homestasis complement previously established actions upon PP2A and the resulting balance between kinase and phosphatase activity in controlling cardiac ion channel activity and rhythmic Ca2+ cycling.In summary, as illustrated in Figure cko mice (Liu et al., 2+ homeostasis and Ca2+ handling in turn regulating cardiomyocyte excitability and contractility (Ke et al., 2+ handling in cardiac disease conditions.Earlier reports (Liu et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RUNX1 at chromosome 21q22 encodes the alpha subunit of Core binding factor (CBF), a heterodimeric transcription factor involved in the development of normal hematopoiesis. Translocations of RUNX1 are seen in several types of leukemia with at least 21 identified partner genes. The cryptic t rearrangement involving the USP42 gene appears to be a specific and recurrent cytogenetic abnormality. Eight of the 9 cases identified in the literature with this translocation were associated with acute myeloid leukemia (AML), with the remaining case showing refractory anemia with excess blasts, type 2. Herein, we present a patient with two preceding years of leukopenia and one year of anemia prior to the diagnosis of AML, NOS with monocytic differentiation (myelomonocytic leukemia) whose conventional cytogenetics showed an abnormal clone with 5q deletion. Interphase FISH using LSI RUNX1/RUNXT1 showed three signals for RUNX1. FISH studies on previously G-banded metaphases showed the extra RUNX1 signal on the short arm of chromosome 7. Further characterization using the subtelomeric 7p probe showed a cryptic 7;21 translocation. Our case and eight previously reported leukemic cases with the t appear to share similar features including monocytic differentiation, immunophenotypic aberrancies (often with CD56 and/or CD7), and a generally poor response to standard induction chemotherapy. About 80% of these cases had loss of 5q material as an additional abnormality at initial diagnosis or relapse. These findings suggest that t may represent a distinct recurrent cytogenetic abnormality associated with AML. The association between the t and 5q aberrancies appears to be non-random, however the pathogenetic connection remains unclear. Additional studies to evaluate for RUNX1 partner genes may be considered for AML patients with RUNX1 rearrangement and 5q abnormalities; however knowledge of the prognostic implications of this rearrangement is still limited.The gene  RUNX1), also known as acute myeloid leukemia 1 protein (AML1) /46,XX[15] .The 1 Figure . FISH st7 Figure . FurtherRUNX1 gene and the loss of chromosome 5q in an AML patient. First reported by Paulsson et al. in AML [In this study, we describe a new case of semi-cryptic t7;21) involving the . in AML , only 9 . in AML -12. Lite. in AML . The incWe compared our case with the other reported t7;21) cases CD56 expression is seen in about 25% ,16 of AMRPS14), which is essential for the maintenance of normal erythroid progenitor cells [RPS14[RPS14, however, this speculation would need to be validated by further molecular testing.One of the genes located on chromosome 5q is ribosomal protein S14 . The high incidence of t found together with del(5q) suggest that this combination is a non-random abnormality. Many questions need to be answered, including: Why does this combination happen with such a high frequency? What is the etiology? What is the significance of these aberrancies in relation to AML? Although a recent large scale cooperative study of 514 MDS patients showed that 5q deletion with additional cytogenetic abnormalities is a marker of poor prognosis , additioRUNX1-USP42 fusion protein. It is reasonable to assume that our case also has the associated RUNX1-USP42 fusion protein, although we did not pursue the exact partner of RUNX1 in this report due to the limited specimen. RUNX1 is a vital regulator of hemopoiesis [RUNX1[RUNX1 translocation is a recurrent feature in many AML and MDS patients, and numerous translocation partners have been described. RUNX1 rearrangment’s leukemogenicity lies in the retention of RUNX1’s DNA binding “Runt homology domain” (RHD) domain, and associated loss of its transactivation domain. The resultant fusion protein acts as a dominant negative inhibitor of wild-type RUNX1 in transcription activation [All previous reports about the t7;21)p22;q22) rearrangement have detected a ;q22 reartivation ,22.RUNX1, ubiquitin specific peptidase 42 (USP42) may also play an important leukemogenic role in t cases. USP42, belonging to the ubiquitin-proteasome system (UPS), is a deubiquitinating enzyme (DUB). The process of ubiquitination and deubiquitination decides the fate of many proteins, many of which are related with cell cycle regulation, DNA repair, and differentiation. USP and the DUBs contain many potential drug targets for the treatment. USP42 itself was reported to increase the stability of p53[RUNX1-USP42 fusion protein loses wild type USP42 function and decreases the stability of p53. Further studies to characterize the involvement of USP42 in our case were not possible due to insufficient sample.While most studies focus on ty of p53. We propIn conclusion, we report a new case of AML with a cryptic t and chromosome 5q abnormality. To the best of our knowledge, this represents one of the few AML patients with this genetic aberrancy. Review of the literature showed that our case shares many common features with other reported cases, which suggests that t may represent a new distinct entity in AML.Consent for publication of this case report and any accompanying images was obtained from the patient.The authors declare that they have no competing interests.JJ and EL contributed equally to get clinical information, flow cytometry and morphological data as well as the cytogenetic and molecular cytogenetic data. LY did all the bench work in this case as well the interpretation of the results. CAT drafted and checked all the manuscript and this case presentation. All authors read and approved the final manuscript.