Rabbit Haemorrhagic Disease (RHD) is caused by a calicivirus (RHDV) that kills 90% of infected adult European rabbits within 3 days. Remarkably, young rabbits are resistant to RHD. We induced immunosuppression in young rabbits by treatment with methylprednisolone acetate (MPA) and challenged the animals with RHDV by intramuscular injection. All of these young rabbits died within 3 days of infection due to fulminant hepatitis, presenting a large number of RHDV-positive dead or apoptotic hepatocytes, and a significant seric increase in cytokines, features that are similar to those of naïve adult rabbits infected by RHDV. We conclude that MPA-induced immunosuppression abrogates the resistance of young rabbits to RHD, indicating that there are differences in the innate immune system between young and adult rabbits that contribute to their distinct resistance/susceptibility to RHDV infection. Oryctolagus cuniculus are the natural hosts of the Rabbit Haemorrhagic Disease Virus (RHDV). This virus targets the liver of rabbits and causes the death of millions of wild and domestic adult rabbits worldwide [Rabbits of the species orldwide -3. Usualorldwide . A centrorldwide  reportedorldwide . They alorldwide ,8, whichorldwide ,9-12. Thorldwide -6,13-16.Oryctolagus cuniculus) were used when the animals were 4 weeks old. The rabbits were kept under standard conditions of housing with unrestricted access to food and water; this was done according to the European Union Directive no. 2010/63/CE and was approved by the appropriate local ethic committee (“Comité de Ética do ICBAS/UP”). The inoculum of RHDV (Ast/89 strain) was obtained as previously described by Teixeira et al. [10 titre in haemagglutination units (HAU). Fourteen young rabbits (24 days) were immunosuppressed with an intramuscular injection of a suspension of MPA a long-acting corticosteroid . All animals were also injected subcutaneously with a broad spectrum antibiotic, enrofloxacine , in order to prevent secondary bacterial infections. One week later, 9 of the 14 immunosuppressed rabbits were challenged with 100 μL of RHDV injected intramuscularly. The other 5 young rabbits were used as controls of the immunosuppression, i.e. they were euthanized and their thymuses were collected and weighed in relation with the body weight of the rabbits [405nm positive control sample; INGEZIM RHDV 17.RHD.K2 kit, Ingenasa, Madrid, Spain). Other liver samples were fixed by 10% buffered formalin, dehydrated through graded alcohols and xylene and embedded in paraffin wax by standard methods. These were used for light microscopy studies that included immunocytochemistry for RHDV antigen , the prs et al. ) and TUNn = 2; 36–48 h n = 6; 60–72 h n = 1) depicting the hyperacute form of RHD, i.e., they died suddenly, often without conspicuous signs of disease, showing prostration, lethargy and anorexia. As expected, all non-immunosuppressed infected young rabbits showed no clinical symptoms of disease after being infected with RHDV; they were euthanized 1 week after the viral inoculation. Immunosuppressed rabbits (infected (IS + I) or not (IS)) showed atrophic thymuses, indicating that immunosuppression had been achieved; this atrophy was confirmed by the ratio thymus (g)/body weight (g) since we found that these ratios were significantly lower in immunosuppressed groups ; p < 0.0001) than in control rabbits . The immunosuppressed, RHDV-infected young rabbits showed the typical lesions of RHD, such as enlarged and pale livers with enhanced lobular patterns, congestion and haemorrhagic lesions of several viscera, and bloody foam in the trachea. A significant difference was seen in the spleen volume, with splenomegaly in immunosuppressed, RHDV-infected rabbits in contrast with spleen atrophy in immunosuppressed animals (ratio - spleen (g)/body weight (g); p < 0.05). Young rabbits that were infected with RHDV did not present any macroscopic lesions suggestive of RHD, 7 days after the viral inoculation, when they were euthanized. Widespread liver cell vacuolization was seen in immunosuppressed, RHDV-infected young rabbits; these hepatocytes often showed pyknotic nuclei and karyorrhexis, classic features of cell death . In contrast, the virus was not detected in the livers of young rabbits that had been submitted to the RHDV infection. The TUNEL assay confirmed marked apoptosis in the thymus of immunosuppressed, RHDV-infected rabbits and this was less extensive in the spleen . The inflammatory infiltrates in the liver of the immunosuppressed, RHDV-infected young rabbits were mainly constituted by heterophils, contrary to what would be expected in infected young rabbits, i.e., where liver infiltrates contain macrophages and lymphocytes ,6. These T cells -25. Our We also report here a marked increase in proinflammatory and anti-inflammatory cytokines  in the serum of the immunosuppressed, RHDV-infected young rabbits. This enhancement in cytokines is similar to what occurs in the blood of infected adult rabbits a few hours prior to death. Others have previously shown that RHDV infection of adult rabbits is related to a strong increase of mRNA levels of pro-inflammatory cytokines in the liver, target organ of viral replication ,27. The In conclusion, we demonstrate that the innate immune response of young rabbits is abrogated by the MPA treatment, changing young rabbits into hosts that become as susceptible to fatal calicivirus infection as naïve adult rabbits are. Thus we propose that the resistance of young rabbits to RHDV-infection is mediated by their particular innate immunity that is different from that of adult rabbits. Moreover, this discovery opens the way for a better understanding of the rationale of resistance of young rabbits to RHD.The authors declare they have no competing interests.RMM designed the study, performed the animal experiments, wrote the manuscript and performed the statistical analysis. LT participated in the animal experiments, helped in the immunohistochemistry analysis and drafting the manuscript. JCR performed the ELISA and TUNEL assays. ACS participated in the animal experiments. APA participated in the study design, interpretation of the results and helped draft the manuscript. PGF contributed to the elaboration of the study design, interpretation of the results and helped draft the manuscript. All authors read and approved the final manuscript.


We and others have demonstrated that contracture and calpain are involved in the Ca2+ paradox-induced injury. This study aimed to elucidate their roles in this model. The Ca2+ paradox was elicited by perfusing isolated rat hearts with Ca2+-free KH media for 3 min or 5 min followed by 30 min of Ca2+ repletion. The LVDP was measured to reflect contractile function, and the LVEDP was measured to indicate contracture. TTC staining and the quantification of LDH release were used to define cell death. Calpain activity and troponin I release were measured after Ca2+ repletion. Ca2+ repletion of the once 3-min Ca2+ depleted hearts resulted in almost no viable tissues and the disappearance of contractile function. Compared to the effects of the calpain inhibitor MDL28170, KB-R7943, an inhibitor of the Na+/Ca2+ exchanger, reduced the LVEDP level to a greater extent, which was well correlated with improved contractile function recovery and tissue survival. The depletion of Ca2+ for 5 min had the same effects on injury as the 3-min Ca2+ depletion, except that the LVEDP in the 5-min Ca2+ depletion group was lower than the level in the 3-min Ca2+ depletion group. KB-R7943 failed to reduce the level of LVEDP, with no improvement in the LVDP recovery in the hearts subjected to the 5-min Ca2+ depletion treatment; however, KB-R7943 preserved its protective effects in surviving tissue. Both KB-R7943 and MDL28170 attenuated the Ca2+ repletion-induced increase in calpain activity in 3 min or 5 min Ca2+ depleted hearts. However, only KB-R7943 reduced the release of troponin I from the Ca2+ paradoxic heart. These results provide evidence suggesting that contracture is the main cause for contractile dysfunction, while activation of calpain mediates cell death in the Ca2+ paradox.The Ca Thirdly, we prolonged the duration of Ca2+ depletion to 5 min to reduce the diminishing effects of KB-R7943 on the LVEDP and then evaluated its effects on the contractile function recovery and survival of tissue after Ca2+ repletion. Finally, we determined the effects of KB-R7943 and MDL28170 on calpain activity and troponin I release. These results suggest that contracture is the main cause for contractile dysfunction, while activation of calpain mediates cell death in the Ca2+ paradox.In this study, KB-R7943, a selective inhibitor of NCX KB-R7943 and MDL28170 were purchased from Tocris Bioscience . The LDH enzyme-linked immunoassay (ELISA) kit was purchased from R&D Systems . Rabbit anti-μ-calpain and -m-calpain antibodies were obtained from Cell Signaling Technology . Mouse anti-α-fodrin antibody was purchased from Enzo Life Sciences . A tetramethylrhodamine goat anti-rabbit IgG was purchased from Molecular Probes . Triphenyltetrazolium chloride (TTC), 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI), Ponceau Red, and other chemicals were from Sigma .Sprague-Dawley male rats of 250∼300 g body weight were obtained from the Laboratory Animal Centers at The Fourth Military Medical University. All experimental procedures described here were in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at The Fourth Military Medical University.4, 1.2 mM KH2PO4, 1.25 mM CaCl2, 25 mM NaHCO3, and 11 mM glucose . The solution was equilibrated with 95% O2/5% CO2. A water-filled latex balloon-tipped catheter was carefully placed into the left ventricle through the left atrium and adjusted to a left ventricular end-diastolic pressure (LVEDP) of approximately 5 mm Hg by inflating the balloon. The distal portion of the catheter was connected to a computer via a transducer . Data processing was performed using AcqKnowledge software (version 3.8.1). The index of myocardial contractile function was determined with the left ventricular developed pressure (LVDP), which was calculated from the difference between the left ventricular peak systolic pressure and end-diastolic pressure. The LVEDP was used to reflect the diastolic function, or the degree of contracture occurring at the end of the experiment. A 10-min equilibration period was allowed before the experiments started.As described previously 2+ depletion protocol, the heart was successively perfused with Ca2+-free KH solution for 3 min and KH solution containing 1.25 mM Ca2+ for 30 min without (Group 4) or with KB-R7943 (Group 5) and MDL28170 (Group 6). In the 5-min Ca2+ depletion protocol, the heart was depleted of Ca2+ for 5 min without (Group 7) or with KB-R7943 (Group 8) and MDL28170 (Group 9).The experiments were randomly divided into 9 groups. Every group had 6 hearts. For the normal control (Group 1), the hearts were perfused for 45 min with normal KH solution. For the drug control (Group 2 and 3), the hearts were treated with 10 µM KB-R7943 or MDL-28170 for 8 min followed by perfusion with normal KH solution. In the 3-min Ca2+-free KH solution, Ca2+ was omitted, and 0.1 mM EDTA was added to ensure the removal of any contaminating Ca2+. A 10 µM concentration of KB-R7943 or MDL28170 was added during the 1-min perfusion prior to Ca2+ depletion, during the entire period of Ca2+ depletion, and for the first 2 min of Ca2+ repletion. The concentrations of KB-R7943 and MDL28170 used in this study were based on previous studies For the CaAt the end of experiments, the heart was frozen, cut into slices perpendicular to the apex-base axis, incubated in 1.0% TTC for 20 min at 37°C, and then immersed in 10% formalin overnight. Viable myocardium was stained brick red and the absence of TTC staining demarcated the areas of irreversibly injured myocardium. The area of myocardial injury was determined by a computerized planimetry technique  and expressed as a percentage of the total area 2+ repletion, and LDH, an indicator of cell death 2+ repletion was normalized against the wet weight of the heart and expressed as IU/g.The coronary effluent was collected during the 30 min of CaCardiac troponin I in the coronary effluent was measured with the current version of the AccuTnI assay . The Access Immunoassay System is a random access, bench-top analyzer that has been described in detail in previous studies The frozen left ventricle tissue was homogenized in an ice-cold Tris-buffered saline solution containing 20 mM Tris-HCl (pH 7.3), 1 mM EGTA, 150 mM NaCl, and 1% Triton X-100 and centrifuged for 15 min at 15,000×g. The supernatant was collected and stored at −70°C until use. Calpain activity was measured by fluorometry (BioTek Synergy HT Microplate Reader) using Suc-LLVY-AMC  as a substrate, as previously described 2+ repletion, the heart was removed, and the left ventricular myocardium was frozen in liquid nitrogen and stored at −70°C until use. Approximately 100 mg of frozen tissue was homogenized, using a Bio-Gen PRO200 homogenizer, for 30 s at 4°C in 0.5 ml of lysis buffer containing 50 mM Tris-HCl (pH 7.3), 150 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, with 1% Triton X-100 and 1% protease inhibitor cocktail. The lysates were centrifuged for 15 min at 12,000×g, and the resulting supernatant was aliquoted and stored at −70°C. Total protein concentrations were determined using the Bradford protein assay kit, and 40 µg of the protein was used to evaluate the calpain activity by Western blot using a monoclonal antibody against α-fodrin, which recognizes the calpain-cleaved fragment (approximately 150 kDa), as described previously Two minutes after Ca2+ repletion, the left ventricle tissue was excised, fixed in 4% paraformaldehyde at 4°C overnight, embedded in paraffin, and cut into 3-µm-thick sections. After deparaffinization and rehydration, the sections were stained with anti-μ-calpain (1∶100) and anti-m-calpain antibody (1∶25) at 4°C overnight, followed by a 60-min incubation at room temperature with a TRITC-labeled goat anti-rabbit IgG second antibody. DAPI was used to label the nuclei. The sections were examined using a laser-scanning confocal microscope equipped with the FV10-ASW system (Olympus FV1000). The images were analyzed using Image-Pro Plus 5.0 software  to quantify the percentage of membrane-anchored calpain, as described previously Two minutes after CaP<0.05 was considered statistically significant.All data are presented as the mean 2+ depletion resulted in no viable tissue, as shown by TTC staining, and an increase in LDH release compared with the control group  After prolonging the duration of Ca2+ depletion to 5 min, KB-R7943 failed to decrease the level of LVEDP and rescue contractile dysfunction, but the treatment still preserved surviving tissue against Ca2+ repletion and inhibited calpain activity. These results provide, for the first time, evidence suggesting that contracture is the main cause for contractile dysfunction, and activation of calpain mediates cell death in the Ca2+ paradox.It is generally accepted that the development of the contracture is the primary event in the Ca2+ depletion treatment, which was accompanied with a reduction in the spike in calpain activity and the degradation of α-fordin. These results support the notion that the activation of calpain is a critical event leading to tissue death in the Ca2+ paradox. Furthermore, our data, consistent with previous studies, identified membrane-anchored μ- and m-calpain in the Ca2+ paradoxical hearts, suggesting that this step triggers a strong activation of this protease 2+ paradox 2+ paradox because mitochondria controls the production of high energy phosphate and cell death, which are regulated by Ca2+In the current study, we found that both KB-R7943 and MDL28170 improved the survival of heart tissue from the 3-min and 5-min Ca2+ paradox include the following major characteristics: 1) abnormalities in the sarcomere structure, i.e., some degree of A-band compression and apparent thickening of the Z-bands; 2) contracture force development, which is characterized by an increase in the LVEDP performance and consequently disruption of the sarcolemmal membrane 2+ depletion group is weaker than the contracture intensity in the 3-min Ca2+ depletion group. This lesser intensity may be due to the amount of Ca2+ in the sarcoplasmic reticulum is decreased by the prolonged Ca2+-free superfusion time 2+ paradox. Compared to MDL 28710, an inhibitor of calpain, KB-R7943 had greatly decreased the level of LVEDP and improved cell survival. We interpret from our results that force stress from the contracture and the activation of calpain are two key nodes in the Ca2+ paradox cascade, which results in hearts injury.Contracture developing in the Ca2+ depletion, which was accompanied with a decreased release of troponin I, a regulatory protein involved in maintaining the diastolic state 2+ influx 2+ overload in the Ca2+ paradox 2+ paradoxic hearts. KB-R7943 failed to rescue heart contractile dysfunction after the 5-min Ca2+ depletion treatment, compared to the corresponding data in the 3-min Ca2+ depletion group. The data measuring the level of troponin I in the coronary effluent showed that, although KB-R7943 reduced the release of troponin I in the Ca2+ paradoxic hearts, the amount of troponin I release in the 5-min Ca2+ depletion with KB-R7943 group was still higher than the amount released in the control hearts (2+ depletion group. In addition, we cannot rule out the possibility that other calcium channels, i.e., the canonical transient receptor potential channels 2+ overload in the Ca2+ paradox.An interesting observation in this study is that KB-R7943 rescued contractile dysfunction after the 3-min Cal hearts . This ma2+ paradoxic hearts, supporting the notion that activation of calpain mediates tissue death, but not contractile dysfunction. In addition, the ubiquitin proteasome system has been reported to mediate sarcomere degradation 2+ overload-induced troponin I release. 2+ depletion with MDL28170 group was smaller than the area in the corresponding 3-min Ca2+ depletion group. We suggest that the disruption of the sarcolemmal membrane caused by contracture aggravates calpain-mediated tissue death.In this study, we found that MDL28170 did not affect the release of troponin I from the Ca2+ homeostasis and the subsequent development of contractures are some of the important factors for diastolic dysfunction 2+ overload has been implicated as an target for treating myocardial ischemia/reperfusion injury These results are clinically relevant because left ventricular diastolic dysfunction with a normal ejection fraction currently represents 40% to 50% of all heart failure cases 2+ paradox. Further studies should focus on unfolding the molecular mechanism underlying the development of the contracture in the Ca2+ paradox.In summary, the present study provides the first evidence suggesting that the contracture is the main cause for contractile dysfunction, while activation of calpain mediates tissue death in the Ca


However, carbenoxolone may directly inhibit light-evoked Ca2+ signals in ipRGCs independent of gap junction blockade.Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). These ganglion cell photoreceptors send axons to several central targets involved in a variety of functions. Within the retina ipRGCs provide excitatory drive to dopaminergic amacrine cells via glutamatergic signals and ipRGCs are coupled to wide-field GABAergic amacrine cells via gap junctions. However, the extent to which ipRGCs are coupled to other retinal neurons in the ganglion cell layer via gap junctions is unclear. Carbenoxolone, a widely employed gap junction inhibitor, greatly reduces the number of retinal neurons exhibiting non-rod, non-cone mediated light-evoked Ca2+ responses in ipRGCs, the light-evoked rise in intracellular Ca2+ ([Ca2+]i) was examined using fura-2 imaging in isolated rat ipRGCs maintained in short-term culture in the absence and presence of carbenoxolone. Carbenoxolone at 50 and 100 µM concentrations completely abolished the light-evoked rise in [Ca2+]i in isolated ipRGCs. Recovery from carbenoxolone inhibition was variable.To test the possibility that carbenoxolone directly inhibits light-evoked Ca2+]i in isolated mammalian ganglion cell photoreceptors is inhibited by carbenoxolone. Since the light-evoked increase in [Ca2+]i in isolated ipRGCs is almost entirely due to Ca2+ entry via L-type voltage-gated calcium channels and carbenoxolone does not inhibit light-evoked action potential firing in ipRGCs in situ, carbenoxolone may block the light-evoked increase in [Ca2+]i in ipRGCs by blocking L-type voltage-gated Ca2+ channels. The ability of carbenoxolone to block evoked Ca2+ responses must be taken into account when interpreting the effects of this pharmacological agent on retinal or other neuronal circuits, particularly if a change in [Ca2+]i is the output being measured.We demonstrate that the light-evoked rise in [Ca Retinal ganglion cells expressing melanopsin are intrinsically photosensitive (ipRGCs) 2+ in the presence of carbenoxolone, a compound widely used to block gap junctions in vitro for multielectrode recording does not decrease the number of retinal neurons generating light-evoked action potentials Gap junctions/electrical synapses are abundant throughout the plexiform layers of the retina and are diverse in their ultrastructural morphology 2+]i) is by Ca2+ influx through L-type voltage-gated calcium channels (VGCC)  although the extent of inhibition was quite variable among ipRGCs at the intermediate concentrations tested (1 and 10 µM) and complete inhibition of the light-evoked Ca2+ response was observed in at least some cells at all concentrations examined except 100 nM. In the presence of 100 µM carbenoxolone, the concentration typically used to block gap junctions between neurons maintained in vitro2+ response in individual ipRGCs was completely abolished  (To determine the direct effect of the gap junction blocker carbenoxolone on light-induced Ca p<0.01) .2+ response in individual ipRGCs was evaluated by recording three light-evoked Ca2+ responses spaced 15 min apart during carbenoxolone washout. The level of recovery of the light-evoked Ca2+ response was related to the concentration of carbenoxolone tested with greater recovery at the lower concentrations. However, recovery of the light-evoked Ca2+ response was variable; 5 of 20 cells showed complete recovery whereas 8 of the 20 ipRGCs showed no recovery after 45 min  evokes a rise in [Ca2+]i in isolated ipRGCs 2+]i in ipRGCs could result from Ca2+ admitted via NMDA and/or AMPA/kainate-type glutamate receptors and via the activation of VGCC occurring after membrane depolarization and action potential firing resulting from glutamate receptor stimulation, as described for immunopanned conventional RGCs + channels. TTX (1 µM) significantly inhibited the glutamate-evoked rise in [Ca2+]i with responses 43.5±10.5% of baseline   . In addis tested , confirm2+]i   in the absence and presence of verapamil, an L-type VGCC antagonist, that has been shown previously to inhibit light-evoked activation of L-type VGCC in isolated ipRGCs resulting from action potential generation  p<0.01) . These d2+]i is reduced by carbenoxolone. After establishing that cells were light responsive and that they responded to glutamate  with an evoked rise in [Ca2+]i (baseline response), cells were stimulated with light in the absence of carbenoxolone (for a baseline Ca2+ response) followed by light in the presence of carbenoxolone (100 µM) and then glutamate  in the presence of carbenoxolone. The light-evoked Ca2+ response was again completely blocked by 100 µM carbenoxolone,   (Lastly we asked whether the glutamate-evoked rise in [Ca t-test)  confirmi p<0.01) .2+ response despite their lack of an increase in [Ca2+]i in response to light stimulation. This finding validates the observed inhibition of the light-evoked Ca2+ response in the absence of recovery from carbenoxolone in ipRGCs in the initial experiment above. Comparison of the data from the initial experiment of 100 µM carbenoxolone inhibition of light-evoked Ca2+ responses (n = 5) with this last experiment examining 100 µM carbenoxolone inhibition of light-evoked Ca2+ responses (n = 7) revealed no significant difference in the level of carbenoxolone inhibition . It can be concluded that 100 µM carbenoxolone virtually eliminates the light-evoked elevation in [Ca2+]i in isolated ipRGCs. In addition, comparison of the glutamate-evoked Ca2+ responses in the presence of TTX, verapamil, or carbenoxolone revealed no significant difference in the inhibitory effect of the three compounds on the level of the glutamate-evoked elevation of [Ca2+]i .These latter data indicate that in the presence of 100 µM carbenoxolone, ipRGCs remained viable and were capable of generating a glutamate-evoked Ca2+]i in solitary, isolated mammalian retinal ganglion cell photoreceptors maintained in short-term culture. Importantly, carbenoxolone blocked the light-evoked Ca2+ response by acting directly on the ipRGCs as these neurons lacked any physical contact with other ipRGCs or other retinal cells in culture. The effects of carbenoxolone on evoked-Ca2+ responses in the present study were obtained by stimulating isolated, individual mammalian retinal neuron photoreceptors with light, coupled with non-invasive calcium imaging. Our findings are in agreement with those of Vessey and colleagues 2+ as a charge carrier) obtained by depolarizing voltage-clamped amphibian cone photoreceptors using whole-cell patch-clamp procedures. The data are consistent with the interpretation that carbenoxolone directly blocks L-type voltage-gated calcium channels. Although the possibility that carbenoxolone may have stimulated the release of unknown tropic factors from cells in the cultures that subsequently inhibited the evoked Ca2+ responses can not be entirely eliminated, it is clear that the effect of carbenoxolone on the light-evoked Ca2+ response in the present study was not the result of carbenoxolone blocking gap junctions between ganglion cell photoreceptors.The principal novel finding in this study is the demonstration that carbenoxolone, a widely used agent for inhibiting neuronal gap junctions, abolished the light-evoked elevation in i in ipRGCs, in addition to blocking gap junction coupling between retinal cells. This direct action of carbenoxolone on light-evoked Ca2+ responses in ipRGCs would have resulted in Sekaran and colleagues greatly overestimating the extent of coupling between ipRGCs and other cells in the ganglion cell layer of the retina 2+ responses in the ganglion cell layer reported by Sekaran and colleagues 2+ responses in cells in the ganglion cell layer described by Sekaran and co-workers 2+ responses in ipRGCs in the intact retina was simply incomplete due to diffusion barriers It was first reported that ipRGCs were extensively coupled to other cells in the ganglion cell layer of the retina via gap junctions based on the reduction in the number of cells demonstrating a light-evoked Ca2+ response in ipRGCs was due to the blockade of L-type VGCC is based on several observations. The primary source for light-evoked elevations in ipRGC [Ca2+]i is via L-type VGCC after light-induced depolarization and action potential firing 2+ in ipRGCs occurs after photon capture by melanopsin and membrane depolarization which is believed to be mediated via canonical transient receptor potential (TRP) channels 2+ by preventing the ipRGCs from depolarizing and firing action potentials. However, carbenoxolone did not alter light-evoked action potential generation in ipRGCs The interpretation that the inhibitory effect of carbenoxolone on the light-evoked Ca2+]i in ipRGCs described in this study. Glutamate acting via glutamate receptors on ipRGCs produces membrane depolarization followed by action potential generation and activation of L-type VGCC; the glutamate evoked rise in Ca2+ was significantly reduced by TTX which blocks action potential generation. The residual Ca2+ response in the presence of TTX was from Ca2+ entry via NMDA and/or AMPA/kainate-type glutamate receptors which are permeable to Ca2+. Thus, blocking glutamate-evoked action potentials eliminates activation of L-type VGCC. Indeed, we next demonstrated that verapamil, a selective L-type VGCC antagonist, inhibited the glutamate-evoked rise in Ca2+ to the same degree as blockade by TTX. Thus if carbenoxolone blocked L-type VGCC, it would be predicted that carbenoxolone would produce a similar level of antagonism as TTX and verapamil; the carbenoxolone inhibition of the glutamate-evoked elevation in [Ca2+]i in ipRGCs was not significantly different from that observed with TTX or verapamil. Taken together, the data strongly suggest that carbenoxolone inhibits L-type VGCC.Additional evidence in support of the interpretation that carbenoxolone blocks L-type VGCC comes from the glutamate-evoked elevation in [Ca2+]i were completely blocked in individual immunopanned ipRGCs, and the effect was not easily reversible . Thus, inhibition of VGCC should also be considered when using meclofenamic acid as a gap junction blocker.Although carbenoxolone is a widely used gap junction inhibitor, the effects of carbenoxolone are difficult and at times impossible to reverse, as reported here and as described in the literature. Pharmacological agents that block gap junctions and are potentially more easily reversible, such as the fenamates, have been sought 2+ responses are the outcome measure or when attempting to assign a role for gap junctions in complex physiological network responses such as those that occur in the retina.In summary, carbenoxolone, a water-soluble membrane permeant pharmacological agent derived from licorice root, inhibits gap junctions but also has actions on other membrane channels including VGCC. Thus it is not a compound that can be used reliably when CaThe use of animals in this study was approved by the University of Nebraska-Lincoln Institutional Animal Care and Use Committee (ID 391) and all experiments were conducted according to NIH guidelines in Principles of Laboratory Animal Care.2+/Mg2+-free Dulbecco's phosphate-buffered saline [DPBS] with papain and DNase) and mechanical trituration; melanopsin-expressing RGCs were isolated using a melanopsin-antibody panning protocol with a rabbit melanopsin antibody (F7884) generated against the N-terminal 19 amino acid sequence of rat melanopsin. Melanopsin-panned cells were plated into wells containing a poly-D-lysine/laminin-coated Biocoat glass coverslip and 750 µl of culture medium consisting of Neurobasal-A plus 2% B27 supplements, 1 mM glutamine, 25 ng/ml BDNF, 10 ng/ml CNTF, 5 µM forskolin, and 10 µg/ml gentamicin. Cultures were maintained in the dark at 37°C in a humidified 5% CO2-air atmosphere.Isolation of melanopsin-expressing ipRGCs was performed as previously described 2+-free, pH 7.4) containing 5 µM fura-2 AM calcium indicator dye for 30 min in the dark at 35°C. The fura-loaded cells were then transferred to a microscope chamber that was constantly superfused with HBSS warmed to 33–35°C and bubbled with 100% oxygen. All tested drugs were dissolved in the HBSS and delivered to the chamber by a peristaltic pump at a rate of ≈1 ml/min.After 1–2 days in culture, the coverslip-plated melanopsin-panned cells were transferred to modified Hank's balanced salt solution  were used to generate fura-2 fluorescence. A filter wheel alternated the 340 and 380 nm filters with a 400 ms excitation period for each wavelength. Fluorescence images (12-bit) were captured with a cooled charged-coupled device camera fitted to an Axioskop 2 FS upright microscope using a water-immersion objective . The images of RGC fluorescence at 340 and 380 nm excitation were calculated over a large area of the RGC soma and converted to ratiometric (340/380 nm) images by imaging software . The background fluorescence was measured from a region on the coverslip devoid of RGCs and subtracted from each image.To minimize the intensity of the fura-2 excitation wavelengths , 8 X 8 pixel binned images were acquired. With this increased binning, adequate fluorescence was attained after reducing the 340 and 380 nm excitation intensities with −0.5 and −2.5 log neutral density filters, respectively. Light responses were calculated as the Δ fura-2 ratio, the peak minus baseline fura-2 ratio. The baseline response was calculated as the average fura-2 ratio of the 3 ratios acquired prior to light or drug exposure and the peak was the maximum fura-2 ratio observed in the 100 s following light offset or drug termination.13 photons/s/cm at 420 nm, 6.9×1013 photons/s/cm at 480 nm, 1.7×1014 photons/s/cm at 540 nm and 3.3×1014 photons/s/cm at 600 nm.The isolated cells were stimulated with broad-spectrum light from a 100 W halogen bulb, with light passing through the condenser underneath the microscope chamber. One minute light stimuli were used and the halogen light was blocked every 10 s to allow for the acquisition of fura-2 images. Light stimulation was remotely triggered using an electronic shutter. The standard light stimulus utilized in the experiments was measured as approximately 2.0×10


Of multiple pathways allowing such entry, the mitochondrial Ca2+ uniporter is a highly Ca2+-selective channel complex encoded by several recently-discovered genes. However, the identity of the pore-forming subunit remains to be established, since knockdown of all the candidate uniporter genes inhibit Ca2+ uptake in imaging assays, and reconstitution experiments have been equivocal. To definitively identify the channel, we use whole-mitoplast voltage-clamping, the technique that originally established the uniporter as a Ca2+ channel. We show that RNAi-mediated knockdown of the mitochondrial calcium uniporter (MCU) gene reduces mitochondrial Ca2+ current (IMiCa), whereas overexpression increases it. Additionally, a classic feature of IMiCa, its sensitivity to ruthenium red inhibition, can be abolished by a point mutation in the putative pore domain without altering current magnitude. These analyses establish that MCU encodes the pore-forming subunit of the uniporter channel.Mitochondrial calcium —codes for the protein subunit that creates the pore.Although several calcium import mechanisms exist, the best-studied pathway involves a pore-forming protein complex called the mitochondrial calcium uniporter. This ion channel has an exquisite selectivity, allowing only calcium into mitochondria even when other ions outnumber it a million-fold. Previously, researchers had identified several genes that are required for the formation of the uniporter, but it had not been established which of these encodes the central pore through which the calcium ions pass. Now, Chaudhuri et al. have shown that one of these—a gene called MCU gene reduced the flow of calcium ions through the uniporter, whereas increasing MCU expression increased calcium transport.Prior studies used optical methods or purified proteins to study genes encoding the uniporter complex, producing controversial results regarding pore identity. This study uses a much more direct assay, namely electrophysiology performed on mitochondrial inner membranes. To access the inner membrane, the authors stripped off the outer membrane from whole mitochondria, and made them expand. By using a technique called voltage-clamping, Chaudhuri et al. were able to precisely measure calcium ion movement through intact or mutated channels. This technique controls confounding factors and minimizes the effect of contaminants that can plague interpretation of data acquired by other methods. They showed that blocking the expression of the MCU gene encodes the pore-forming subunit of the channel complex—they identified a single point mutation in MCU that did not affect the channel’s ability to transport calcium ions, but did abolish its sensitivity to ruthenium red. Together, these results show that the MCU gene encodes the pore of the mitochondrial calcium uniporter, and should lead to further research into the physiology and structure of this channel.One unique feature of the mitochondrial calcium uniporter is that it can be blocked by a dye called ruthenium red. Chaudhuri et al. used this property to confirm that the DOI:http://dx.doi.org/10.7554/eLife.00704.002 So far, the discovery of each of these genes occurred via Ca2+ imaging assays following RNAi-mediated knockdown. Although extremely useful for screening, this technique is ill-suited for separating modulatory effects from altered expression of the channel-forming subunit itself, and each of these genes was found to inhibit Ca2+ uptake via this assay. This difficulty arises because Ca2+ imaging measures Ca2+ entry, which depends critically on (i) the number of open portals, (ii) the voltage gradient, and (iii) the Ca2+ concentration gradient driving flux, and none of these is controlled during imaging experiments. In fact, changes in Ca2+ buffers or efflux pathways can alter matrix Ca2+, and changes in pH or other divalents can alter indicator fluorescence, without reflecting a true difference in uniporter activity. Rather, the ideal method for assaying electrogenic ion transport is voltage-clamping, which allows precise control of the voltage and concentration gradient and thus accurate measurement of the changes in channel density.These discoveries have spurred an explosion of research into mitochondrial CaMCU, which possesses two transmembrane domains and highly-conserved acidic residues in a putative pore domain  for altered key features. Using this method, we show here that MCU does recapitulate key features of IMiCa, and show that a single point-mutation can generate resistance to pharmacologic inhibition.To overcome the limitations of single-channel recordings, we chose to voltage-clamp whole mitoplasts  . This alMCU produced a substantial reduction in the protein when assayed by Western blot  of 20 mM [Ca2+]bath, we maximized current by recording in a 100 mM Ca2+ gluconate bath solution , was consistent across all conditions tested here .Compared to the control condition,  smaller . The RuRp<0.001, , with noIMiCa. We focused on an MCU mutated to encode an alanine at a highly-conserved serine in the putative pore-forming loop (S259A)  . The wil (S259A) . To conf (S259A) . Since t (S259A) , we founy to RuR .10.7554/IMiCa. In Ca2+-imaging experiments, this mutant had preserved Ca2+ uptake but diminished sensitivity to uniporter inhibitors , and to determine if the genetically-modified channels could alter them.In our study, we use the most direct assay available to measure mitochondrial Caysiology . By isolMCU transcripts via knockdown, or enhancing them through overexpression, produce parallel changes in IMiCa. Second, we show that a central feature of IMiCa—its exquisite sensitivity to blockade with ruthenium red—can be abolished via a single point mutation in the pore domain. MCU subunits carrying this mutation act in a dominant-negative fashion, producing functional channels that conduct Ca2+ ions but are largely insensitive to ruthenium red. We conclude that the functional uniporter pore at the mitochondrial inner membrane is formed by MCU multimers. We believe these experiments put to rest the outstanding question of the pore identity, firmly establishing that MCU produces IMiCa.We focused on MCU, the leading candidate for the pore-forming subunit, as it possesses two transmembrane domains and has highly-conserved acidic residues in a putative pore-like domain. First, we show that reducing MCU. 2 days after infection, cells were split and selected with the appropriate antibiotic. Stable HEK-293T cells lines expressing shGFP and shMCU were maintained in 2 μg/ml puromycin , while MCU-FLAG and S259A lines were maintained in 200 μg/ml hygromycin B (Sigma).HEK-293T were obtained from ATCC and grown in Dulbecco’s modified Eagle medium with high glucose supplemented with 10% fetal bovine serum and penicillin/streptomycin . HEK-293T cells  were infected with lentivirus expressing short hairpin RNA (for knockdown experiments) or wild-type or mutant FLAG-tagged MCU, we used a hairpin targeting the 3′ UTR, with sequence 5′-GCAAGGAGTTTCTTTCTCTTT-3′ (TRCN0000133861). The control hairpin was against GFP, with sequence 5′-ACAACAGCCACAACGTCTATA-3′ (shGFP) (TRCN0000072181). The FLAG-tagged full-length human MCU cDNA (NM_138357.1) was cloned into the pLJM5 vector . Human Mitofusin-1 (NM_033540) was cloned out of HEK-293T cell cDNA  and ligated in-frame into the pEGFP-N3 vector  using KpnI and BamHI restriction sites. Matrix-targeted mCherry was engineered by replacing the GFP construct of the pAcGFP1-Mito plasmid (Clontech), using BamHI and NotI restriction sites.Vectors for expressing shRNA (pLKO.1) were obtained from the Broad Institute’s RNAi Consortium (Sigma). To silence 5 vector . The MCUMCU were designed using PRIMER-Blast (NCBI). The forward primer was 5′-TTCCTGGGACATCATGGAGC-3′, and the reverse primer was 5′-TGTCTGTCTCTGGCTTCTGG-3′. For qPCR, confluent HEK-293T cells were resuspended in TriZOL reagent (Life Technologies), and total RNA was subsequently purified following the manufacturer’s instructions. 2 μg of RNA was used to synthesize cDNA using SuperScript ViLo (Life Technologies) and 100 ng of this was used per well for qPCR. Reactions were run in triplicate using SYBR Green (Agilent) on an Eppendorf Mastercycler epgradient S Realplex4 qRT-PCR cycler . Analysis compared expression in shMCU cells to shGFP cells using the 2−ΔΔCt method, normalizing to glyceraldehyde 3-phosphate dehydrogenase expression.Primers against human 10 μg of protein from cell lysates or 5 μg of protein from crude mitochondrial preparations were loaded on 12% or 18% Tris-Glycine gels, proteins were transferred to PVDF membranes and then incubated with the indicated antibodies at 4°C overnight. We used antibodies against TOMM20 , TIMM23 , PPIF , ATP5B , FLAG epitope , and MCU .g for 10 min at 4°C to pellet nuclei and non-lysed cells. The supernatant was removed and spun separately at 8000g for 10 min at 4°C to pellet the mitochondrial fraction. Mitochondria were resuspended in a sucrose buffer  to a final concentration of 1 mg/ml. 20 μg of mitochondria were treated with digitonin  or 1% Triton-X100 (Sigma) and 100 μg/ml of proteinase K for 15 min at room temperature, in a 30 μl final volume. Proteinase K was then inhibited by addition of 5 mM PMSF. 5 μg of protein was loaded to Tris-glycine gels for Western blotting.Two confluent plates of HEK-293T cells were washed with PBS and the cells scraped into 1.5-ml microcentrifuge tubes in 1 ml of final volume. The cells were lysed by passaging through a 27.5 G needle several times. Samples were spun at 800Stably-transfected HEK-293T cells were washed in PBS, which was used as the base solution in all following steps. Cell were fixed in 4% formaldehyde, permeabilized in 0.5% Triton-X100, and blocked in 10% goat serum, 0.1% Tween-20 . Primary antibodies were a rabbit anti-FLAG and a mouse anti-MTCO1 . Secondary antibodies were Alexa-488 conjugated to a goat anti-rabbit, and Alexa-546 conjugated to goat anti-mouse (Life Technologies). Cells were imaged on an Olympus Fluoview 1000 confocal microscope .For mitoplast images, HEK-293T cells were co-transfected with mt-mCherry and MFN1-GFP plasmids using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer’s instructions. Mitoplasts were isolated (as described in ‘Whole-mitoplast recording’) 2 days following transfection, and imaged on the confocal microscope as above.Brightness levels have been slightly adjusted in the images to improve contrast. This adjustment has been applied to each image as a whole and has not obscured or eliminated any particular feature of the image. All reagents were from Sigma unless otherwise stated.g pelleted nuclei. The supernatant was transferred to a new tube and centrifuged at 8500g to pellet a crude mitochondrial fraction. This pellet was resuspended in a hypertonic mannitol solution  for 10 min. The suspension was then passed through a French Pressure Cell homogenizer  at 2000 p.s.i. to disrupt the mitochondrial outer membrane. The eluate was centrifuged at 10,500g to pellet mitoplasts. Mitoplasts were stored in a hypertonic solution  on ice and protected from light.We adapted prior methods to HEK-293T cells . Cells w2+ solution  and high-Ca2+ +100 nM RuR solution (Sigma). RuR solutions were prepared fresh for every experiment. Solution exchanges were rapid, as prolonged matrix exposures to Ca2+ caused activation of a significant leak component. Comparisons were made via the Student’s t-test.For recordings, mitoplasts were aliquoted into the initial bath solution  to allow them to expand for 20 min. An individual mitoplast was visualized and attached to the pipette tip with gentle suction. Pipettes were formed from borosilicate glass  with tip resistances of 20–60 MOhm. We used sodium gluconate for pipette internal solutions . Electrophysiology was performed using an Axopatch 200B amplifier . Upon giga-ohm seal formation, pipette capacitance transients were cancelled, and the inner membrane was ruptured by 10–50 ms pulses (350–1000 mV). After establishing the whole-mitoplast configuration, a series of 4 ms pulses of 20 mV were recorded to allow curve fitting for capacitance measurements. Bath solutions were subsequently exchanged for a high-Ca review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent ; Jon Lederer (peer reviewer).The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.2+ uniporter using the patch–clamp technique. Although indirect optical methods helped to identify three candidate proteins important for the activity of the mitochondrial Ca2+ uniporter , the subsequent attempts to understand the exact functions of these proteins led to little progress and produced controversial results. In this manuscript, the authors provide strong evidence that one of the candidate proteins, MCU, forms the pore of the mitochondrial Ca2+ uniporter. Since this is the first structure–function study of the MCU using the patch–clamp technique, methodologically this works sets an important precedent in the field.This work for the first time addresses structure-function relationships within the mitochondrial Ca2+ uniporter. There are two important points to be addressed:Overall, the manuscript is well written and provides sufficient evidence to suggest that the MCU protein is a pore-forming subunit of the mitochondrial Ca1) The quality of the image of an 8-shaped mitoplast on 2)  1) The quality of the image of an 8-shaped mitoplast on .We have replaced the mitoplast image from For this image, we co-transfected HEK cells with a plasmid encoding a mitochondrial matrix-targeted mCherry fluorescent protein, as well as a plasmid encoding GFP-tagged mitofusin-1, a mitochondrial outer membrane GTPase. This image is free of debris, and demonstrates that the structure is one mitoplast. We have replaced the figure legend as follows:“A HEK-293T cell mitoplast under differential interference contrast (far left), showing a typical figure-eight shape. The lobe bounded only by the mitochondrial inner membrane appears less dense (white arrow) than the lobe also bounded by the outer membrane (black arrow). Matrix-targeted mCherry demonstrates that the inner membrane forms a surface on both lobes (middle left). GFP-tagged mitofusin-1  reveals the outer membrane partially encapsulating only one lobe. Far right: merged image.”2) .We have taken out the mention of I/V curves, and added the following to the figure legend to describe the voltage command protocol:“Voltage ramps from −160 to +80 mV for 750 ms were delivered every 6 seconds from a holding potential of 0 mV.”


It has recently been observed or highlighted in other cell types such as myoblasts and neurons, suggesting a wider physiological role of this pathway. Whereas Orai1 protein is considered to be the channel allowing the SOCE in T cells, it is hypothesized that other proteins like TRPC could associate with Orai1 to form SOCE with different pharmacology and kinetics in other cell types. Unraveling SOCE cell functions requires specific effectors to be identified, just as dihydropyridines were crucial for the study of Ca2+ voltage-gated channels, or spider/snake toxins for other ion channel classes. To identify novel SOCE effectors, we analyzed the effects of 2-aminoethyl diphenylborinate (2-APB) and its analogues. 2-APB is a molecule known to both potentiate and inhibit T cell SOCE, but it is also an effector of TRP channels and endoplasmic reticulum Ca2+-ATPase.Store-Operated Calcium Entry (SOCE) is the major CaA structure-function analysis allowed to discover that the boron-oxygen core present in 2-APB and in the borinate ester analogues is absolutely required for the dual effects on SOCE. Indeed, a 2-APB analogue where the boron-oxygen core is replaced by a carbon-phosphorus core is devoid of potentiating capacity (while retaining inhibition capacity), highlighting the key role of the boron-oxygen core present in borinate esters for the potentiation function. However, dimesityl borinate ester, a 2-APB analogue with a terminal B-OH group showed an efficient inhibitory ability, without any potentiating capacity. The removal or addition of phenyl groups respectively decrease or increase the efficiency of the borinate esters to potentiate and inhibit the SOCE. mRNA expression revealed that Jurkat T cells mainly expressed Orai1, and were the more sensitive to 2-APB modulation of SOCE.This study allows the discovery of new boron-oxygen core containing compounds with the same ability as 2-APB to both potentiate and inhibit the SOCE of different leukocyte cell lines. These compounds could represent new tools to characterize the different types of SOCE and the first step in the development of new immunomodulators. The rate was twofold increased in the presence of 10 μM 2-APB (32.4 ± 7.8 nM/s vs. 62.5 ± 11.5 nM/s). In the presence of 50 μM 2-APB, the Ca2+ influx rate was largely decreased (< 1 nM/s) and the [Ca2+]i increase after CaCl2 adding was largely blunted  in a CaR Figure , and subJurkat T  and chicJurkat T , 2-APB h reduced . During d Figure .2+ ions: as Mn2+ ions enter the cells by the same channels as Ca2+ ions, they can not be pumped back to the external medium by the plasma membrane Ca2+ ATPases (PMCA) and lead to indo-1 fluorescence quenching at 430 nm i variations are increased by 200-250% at 3-10 μM, concentrations without any known effect on TRP channels. As DPBA does not possess an ethanolamine group, this confirms that this group plays no role in potentiation process.DPBA had been previously used and was known as a five fold more potent inhibitor of platelet SOCE than 2-APB , meanin. 10 μM, ,32. In o2+ signalling, is an analogue of 2-APB where the two phenyl groups were replaced by two butyl. On the three cell lines, we found that 2-ABB, like 2-APB and DPBA, has dual effect on Ca2+ influx, meaning that the two phenyl rings are not an absolute requirement for potentiation and inhibition processes. However, 2-ABB is less potent to potentiate AND inhibit SOCE than 2-APB and DPBA. A clear correlation seems to exist between the number of phenyl rings in the molecule and its ability to potentiate/inhibit SOCE: DPBA (4 phenyl groups) > 2-APB (2) > 2-ABB (0). Thus, it seems that the phenyl rings do not play a direct role in potentiation, but rather favour it. We can assess that after the binding of 2-APB to the channel protein by the BOC, the two phenyl rings strengthen the binding by a second interaction. In DPBA, the presence of four phenyl rings reinforces this binding. It was previously shown that addition of a phenyl ring to the 2-APB molecule could increase its inhibitory efficacy on platelet SOCE i (3500 nM), 2-APB is not potentiating. Furthermore, the expression levels of Orai1 and STIM1 have been shown to affect the Ca2+ conductance and the potentiation of the currents by 2-APB i was calculated according to the equation [Ca2+]i = Kd (R-Rmin)/(Rmax-R), where Kd is the apparent dissociation constant of indo-1 for Ca2+ , equipped with a thermostatted (37°C) cuvette holder. Ultraviolet light of 360 nm was used for excitation of indo-1, and emission at 405 and 480 nm were recorded. Background and autofluorescence of the cells were removed from the values measured at 405 and 480 nm. The maximum indo-1 fluorescence , and R , we measure the variation of [Ca2+]i per second during the first 20 s after CaCl2 addition: in these conditions, the activities of the plasma membrane Ca2+ ATPases (PMCA) and of the Na+/Ca2+ exchanger are largely reduced . Thus, the Δ[Ca2+]i is only due to the Ca2+ ion entry i: intracellular Ca2+ concentration; ER: endoplasmic reticulum; TRP: transient receptor potential; BOC: boron-oxygen core; TG: thapsigargin; DPBA: diphenylborinic anhydride; DPTHF: 2,2-diphenyltetrahydrofuran; PBA: phenylborinic acid; DP3A: 3-(diphenylphosphino)-1-propylamine; DMBA: dimesitylborinic acid; 2-ABB: 2-aminoethyl dibutylborinate.SOCE: Store-operated calcium entry; SOC: Store-Operated Channel; CRAC: Calcium-Release Activated Calcium; IP2+]i measurements and Orais expression assays, and drafted the manuscript. PM carried out some [Ca2+]i measurements and compound tests.OD carried out the [CaCC conceived and designed the study. All authors have approved the final manuscript


C. elegans meiosis but is dispensable for later steps of meiotic recombination. DSB-2 localizes to chromatin during the time of DSB formation, and its disappearance coincides with a decline in RAD-51 foci marking early recombination intermediates and precedes appearance of COSA-1 foci marking CO-designated sites. These and other data suggest that DSB-2 and its paralog DSB-1 promote competence for DSB formation. Further, immunofluorescence analyses of wild-type gonads and various meiotic mutants reveal that association of DSB-2 with chromatin is coordinated with multiple distinct aspects of the meiotic program, including the phosphorylation state of nuclear envelope protein SUN-1 and dependence on RAD-50 to load the RAD-51 recombinase at DSB sites. Moreover, association of DSB-2 with chromatin is prolonged in mutants impaired for either DSB formation or formation of downstream CO intermediates. These and other data suggest that association of DSB-2 with chromatin is an indicator of competence for DSB formation, and that cells respond to a deficit of CO-competent recombination intermediates by prolonging the DSB-competent state. In the context of this model, we propose that formation of sufficient CO-competent intermediates engages a negative feedback response that leads to cessation of DSB formation as part of a major coordinated transition in meiotic prophase progression. The proposed negative feedback regulation of DSB formation simultaneously (1) ensures that sufficient DSBs are made to guarantee CO formation and (2) prevents excessive DSB levels that could have deleterious effects.For most organisms, chromosome segregation during meiosis relies on deliberate induction of DNA double-strand breaks (DSBs) and repair of a subset of these DSBs as inter-homolog crossovers (COs). However, timing and levels of DSB formation must be tightly controlled to avoid jeopardizing genome integrity. Here we identify the DSB-2 protein, which is required for efficient DSB formation during  C. elegans DSB-2 as a key regulator of DSB formation, and we propose that its association with chromatin is an indicator of DSB competence. Disappearance of DSB-2 is part of a coordinated transition affecting multiple distinct aspects of the meiotic program, and failure to form crossover-eligible recombination intermediates elicits a delay in DSB-2 removal and other transition events. Our data are consistent with a model in which meiotic DSB formation is governed by a negative feedback network wherein cells detect the presence of downstream crossover intermediates and respond by shutting down DSB formation, thereby ensuring that sufficient DSBs are made to guarantee crossovers while simultaneously minimizing the threat to genomic integrity.Formation of haploid gametes during meiosis relies on deliberate induction of DNA double-strand breaks (DSBs), followed by repair of a subset of DSBs as crossovers between homologous chromosomes. Crossovers form the basis of connections that enable homologs to segregate toward opposite spindle poles at meiosis I, thereby reducing ploidy. Thus, germ cells must generate enough DSBs to guarantee a crossover for every chromosome pair while avoiding an excessive number of DSBs that might endanger their genomes. Here, we provide insight into how this crucial balance is achieved. We identify  For most diploid organisms, the formation of haploid gametes relies on crossover (CO) recombination between homologous chromosomes for accurate chromosome segregation. Recombination is initiated during meiotic prophase by the programmed induction of DNA double strand breaks (DSBs), catalyzed by the evolutionarily conserved topoisomerase-like protein Spo11 DSB formation must be tightly regulated to ensure successful meiosis: cells must both turn on DSB formation to achieve inter-homolog COs, but also turn off DSB formation to allow repair and subsequent chromosome re-organization in preparation for the meiotic divisions. Thus, DSB formation and repair must be coordinated with other aspects of meiotic chromosome dynamics. In addition, cells must make enough DSBs to guarantee one CO per chromosome pair, but too many DSBs could lead to unrepaired DNA damage and compromise genomic integrity.C. elegans), Spo11-dependent DSBs can form independently of Rad50/Mre11 in some contexts C. elegans, the chromatin-associated proteins HIM-17, XND-1, and HIM-5 have been implicated in promoting normal levels and/or timing of DSB formation, particularly on the X chromosomes While Spo11 catalyzes DSB formation, little is known about how Spo11 activity is regulated and how the timing and number of DSBs are controlled. Several proteins besides Spo11 are required for meiotic DSB formation in various systems, although their mode(s) of action are not well understood C. elegans DSB-2 protein  as a novel factor required specifically to promote the DSB step of meiotic recombination. We show that DSB-2 localizes to chromatin in meiotic prophase germ cells, and that the timing of its appearance and disappearance corresponds to the time window during which DSBs are formed. These and other data implicate DSB-2 in regulating the timing of competence for DSB formation by SPO-11. Further, we find that the presence of DSB-2 on chromatin is regulated coordinately with multiple distinct aspects of the meiotic program, including specialized meiotic DSB repair features and the phosphorylation state of nuclear envelope protein SUN-1. Thus, we propose that disappearance of DSB-2 reflects loss of competence for DSB formation, which occurs as part of a major coordinated transition in meiotic prophase progression. Moreover, our data suggest the existence of a regulatory network wherein germ cells can detect the presence or absence of downstream CO-eligible recombination intermediates. In the context of this model, successful formation of monitored intermediates would trigger removal of DSB-2 (and other factors) from chromatin and consequent shut-down of DSB formation, whereas a deficit of relevant intermediates would elicit a delay in DSB-2 removal (and in other aspects of meiotic progression). We propose that the negative feedback property inherent in such a regulatory network provides a means to ensure that sufficient DSBs are made to guarantee CO formation, while at the same time protecting the chromosomes against formation of excessive levels of DSBs that could jeopardize genomic integrity.In the current work, we identify the dsb-2(me96) allele was isolated following EMS mutagenesis in a screen for meiotic abnormalities visible in oocytes at diakinesis, the last stage of meiotic prophase (see dsb-2(me96) mutant exhibit a variable number of unattached (achiasmate) chromosomes , indicating a defect in chiasma formation  phenocopied the me96 mutant  of F26H11.6 , me96 is presumed to be a null allele. A second, independently-isolated, dsb-2 allele (me97) contains a premature stop at codon 168 in the same gene, further confirming the identity of F26H11.6 as dsb-2.The 6 mutant . SequencC. elegans (F08G5.1), and genes encoding orthologs of both proteins were found in other Caenorhabditis species  but were not identified in other organisms  hermaphrodites fixed one day after the L4 larval stage (24 hours post-L4) had an average of 8.5 DAPI-stained bodies at diakinesis , 48 hour post-L4 hermaphrodites had an average of 11.1 DAPI bodies . Further, as lack of chiasmata connecting homologs results in mis-segregation of chromosomes, both the frequency of inviable embryos  and the frequency of males  produced by dsb-2(me96) hermaphrodites likewise increased with maternal age  allele, using GFP::COSA-1 as a cytological marker of crossover (CO) sites (dsb-2(me97) worms at 24 hours post-L4 and further declined by 48 hours post-L4 .Age dependence of the O) sites . During dsb-2 mutants is not caused by persistence of maternal gene product in the germ line, as it was observed in homozygous mutant worms derived from either heterozygous parents or homozygous mutant parents . In addition, the age effect is evident at both standard (20°C), and elevated (25°C) growth temperatures.The age effect in Together, our data indicate that the function of DSB-2 is required throughout reproductive life to generate normal levels of COs and chiasmata, and becomes increasingly important for meiotic success in germ cell nuclei that enter the meiotic program at progressively later times. This implies that changes must occur as the worms age that render crossing over and chiasma formation increasingly sensitive to the loss of DSB-2 protein.C. elegans meiosis dsb-2 worms at 48 hours post-L4, when the CO/chiasma deficit is severe. Several lines of evidence indicate that the lack of chiasmata in dsb-2 mutants is due to a defect in the initiation of meiotic recombination.Successful chiasma formation requires pairing of homologous chromosomes, assembly of the synaptonemal complex (SC), and CO recombination between the homologs. Homolog pairing and SC assembly are not dependent on initiation or progression of recombination during dsb-2 mutant worms are proficient for pairing of the X chromosomes, as immunofluorescence of pachytene nuclei showed a single focus of HIM-8, a protein that binds a specific region of the X chromosome known as the pairing center dsb-2 mutants are proficient for assembly of the SC, as immunostaining revealed proper loading of HIM-3  and SYP-1  dsb-2 mutants are deficient in the process of meiotic recombination per se.First, dsb-2 germ lines display greatly reduced levels of RAD-51 foci, with most nuclei having no foci  by the SPO-11 protein  no foci , suggestadiation .C. elegans, providing DSBs by irradiation rescues chiasma formation in the spo-11 mutant, which lacks the enzyme responsible for making programmed DSBs dsb-2 mutant worms, demonstrating that the chiasma defect in dsb-2 worms is a result of a defect in SPO-11-induced DSB formation. In both dsb-2 worms and age-matched spo-11 worms, 1 kRad of irradiation resulted in efficient restoration of chiasmata in diakinesis-stage oocytes examined 18 hours post-irradiation  appears comparable to age-matched wild-type controls despite that fact that RAD-51 foci are already substantially diminished by this stage; this indicates that the presence of DSB-1 on chromatin is not sufficient to promote efficient DSB formation in the absence of DSB-2. Further, the association of residual DSB-1 protein in the dsb-2 mutant appears to change with age, as DSB-1 staining in older dsb-2 germ lines (48 hours post-L4) is typically fainter and declines and disappears sooner than in WT. Together these data suggest that DSB-2 may be required to augment the DSB-promoting activity of DSB-1, possibly by affecting the nature of its association with chromatin, and that the reliance on DSB-2 for this augmentation becomes more acute with increasing age.Whereas DSB-2 localization is abolished in 2 mutant . DSB-1 sspo-11 mutants, which lack endogenous DSBs, DSB-2 is detected on chromosomes in transition zone and pachytene nuclei, and the overall appearance of the staining within nuclei is similar to that in WT nuclei. However, DSB-2 association with chromatin extends further into late pachytene, suggesting that endogenous DSB formation affects timing of DSB-2 removal  protein SUN-1Co-staining experiments revealed that DSB-2 and SUN-1 S8P tend to be detected in the same nuclei . The relchk-2 mutant gonads  null mutant germ lines despite severe defects in germline organization and abnormal chromosome morphology (data not shown). Thus, these two features appear to be independent downstream readouts of CHK-2 activity in meiosis. Together, our data suggest that CHK-2 coordinates the meiotic program by acting as a common upstream regulator of two parallel pathways, thereby linking competence for DSB formation (mediated through DSB-2) with chromosome and NE dynamics (mediated through SUN-1 S8P).Whereas localization of DSB-2 on chromatin and Ser-8 phosphorylation of SUN-1 at the NE in meiotic prophase nuclei tend to be correlated, they do not depend on each other. SUN-1 S8P immunostaining is present on meiotic prophase nuclei in him-19 mutants, which show an age-dependent pleiotropic phenotype that includes multiple defects  that are hypothesized to result from mis-regulation of CHK-2 activity him-19 worms, SUN-1 S8P is missing from most of the TZ and early pachytene regions, but is present on a few scattered nuclei The correlation between DSB-2 and SUN-1 S8P was also tested in spo-11 and him-17 mutants, the zone of DSB-2 and SUN-1 S8P marked nuclei was extended beyond what was seen in WT , suggesting a model in which removal of DSB-2 (and presumably other factors) results in shutting down of DSB formation. Based on these data, we propose that DSB-2 regulates competence for SPO-11-dependent DSB formation during C. elegans meiosis.In this work, we identify DSB-2 as a protein that is required for efficient meiotic DSB formation and that localizes to chromatin during the stages of meiotic prophase when DSBs are thought to form. DSB-2 localizes to chromatin independently of SPO-11 (and thus of DSB formation) and is restricted to the region of the gonad where RAD-51 foci mark processed DSBs (from TZ to mid-pachytene). Further, the fact that exogenous DSBs induced by irradiation rescue the chiasma defect in C. elegans. Whereas HIM-17, XND-1 and HIM-5 proteins localize to chromatin in nuclei throughout the germ line him-17 and xnd-1 mutants display pleiotropic phenotypes indicating that HIM-17 and XND-1 have additional roles regulating germ line proliferation and/or organization dsb-2 mutants are specifically defective in meiotic DSB formation. In addition, whereas XND-1 and HIM-5 affect DSB formation predominantly on the X chromosomes, DSB-2 is required for efficient DSB formation on all chromosomes. Together these data suggest that DSB-2 has a more direct role in promoting DSB formation than do HIM-17, XND-1 or HIM-5.Several properties distinguish DSB-2 from other previously identified chromatin-associated proteins  that influence DSB formation in We interpret the region of the germ line where nuclei are positive for DSB-2 localization to represent the zone in which nuclei are competent to undergo DSB formation. Consistent with this interpretation, in meiotic mutants in which the DSB-2-positive zone is extended (and that are capable of making DSBs and loading RAD-51), RAD-51 foci are higher in number and persist beyond mid-pachytene him-17 mutants, which are defective in DSB formation, suggests that the patches may have functional significance.How might DSB-2 control DSB competence? Given its broad yet uneven localization on chromatin, it might act by altering chromatin structure to create an environment that is permissive for the activity of SPO-11 and the DSB machinery. It might also act directly upon SPO-11 and the DSB machinery, by recruiting and/or activating it at certain locations depending upon the underlying chromatin structure. It is intriguing that DSB-2 localizes to a few bright patches/foci in addition to its broader chromatin staining. The fact that these bright patches are absent in Immunofluorescence analyses of DSB-2 in both wild type and meiotic mutants were highly informative regarding how DSB formation is coordinated with multiple distinct aspects of the meiotic program. We found that presence of DSB-2 on chromosomes and the presence of SUN-1 S8P are highly correlated, despite the fact that neither feature is required for the other. Further, we identified CHK-2 as a common upstream regulator of these two features, and we suggest that CHK-2 links acquisition of competence for DSB formation (promoted by DSB-2) with nuclear/chromosomal processes required for successful pairing and synapsis of homologous chromosomes (mediated by SUN-1 at the NE). Moreover, the correlated removal of both DSB-2 and SUN-1 S8P at mid-pachytene, at the same time that RAD-51 foci disappear, further suggests the existence of coordinated regulatory mechanisms that shut down competence for DSB formation and change other properties of the nucleus as germ cells transition to a later stage of meiotic progression.C. elegans meiosis.As seen in multiple experimental systems, DSB formation is restricted to a specific time window in early prophase, indicating that cells must have a means to shut down the meiotic DSB machinery spo-11, him-17, rad-50), in mutants with defects in early steps of DSB processing , as well as in mutants that can make DSBs but repair them by pathways that do not yield inter-homolog COs . Although we cannot exclude the possibility that different defects in these mutants elicit the same response, the parsimonious explanation is that DSB-2 persistence reflects a response to the common deficit shared by all of these mutants, i.e., the inability to generate CO recombination intermediates. Thus, we infer that CO-eligible recombination intermediates are required for removal of DSB-2 with WT timing. We propose a model in which the appearance of CO-eligible recombination intermediates results in a signal  that is necessary to trigger the shutdown of DSB formation, in part by removal of DSB-2 (i.e. CO-eligible recombination intermediates) feeds back to inhibit an earlier step in the pathway (i.e. DSB formation). Thus, we envision a regulatory network governing DSB formation that involves negative feedback operating on (at least) two levels, one that inhibits DSB formation locally  likely involves the activities of one or more protein kinases. As the CHK-2 protein kinase is required to promote the acquisition of both DSB-2 and SUN-1 S8P, it is likely that the disappearance of DSB-2 and SUN-1 S8P requires inactivation of CHK-2, suggesting that CHK-2 may be a key target of feedback regulation. Further, DSB-2 contains several potential phosphorylation sites both for CHK-2 and for the ATM/ATR protein kinases We speculate that the regulatory network that coordinates this meiotic transition  e.g., the fluidity of the nuclear membrane, which is modified upon entry into meiotic prophase The fact that DSB-2 and SUN-1 S8P are coordinately removed in wild-type meiosis (and coordinately prolonged in mutants) implies that the NE also responds to signaling from CO-eligible recombination intermediates. Our findings confirm and extend the recent report of Woglar While DSB-2 and SUN-1 S8P immunofluorescence signals become dimmer and disappear from most nuclei by the time they reach the mid-pachytene region of the germ line, a few “outlier” nuclei show bright DSB-2 and SUN-1 S8P staining later in the pachytene region. Sometimes the chromatin in these nuclei has a clustered organization reminiscent of zygotene or early pachytene stages, but in contrast to earlier nuclei, these outlier nuclei have brighter DSB-2 staining covering most of the chromatin as well as high levels of RAD-51 foci. This difference suggests that these nuclei are arrested in their progression and may have triggered a checkpoint response. This response could be due to failure to make appropriate CO-eligible recombination intermediates and/or to the presence of excess or persistent DNA breaks. These processes may be inter-related: if the failure to make CO-eligible recombination intermediates keeps DSB formation active, this could increase the chance of accumulating levels of DNA damage that challenge the capacity for repair. Accumulation of high levels of DSB-2 and SUN-1 S8P may indicate that these nuclei are triggering the recombination/DNA damage checkpoint and will be targeted for future apoptosis. While these outlier nuclei may be destined for apoptosis, however, they likely have not yet engaged the cell death program, as outlier nuclei are still detected in mutants lacking the pro-apoptotic factors CED-3 or CED-4 dsb-2 mutant phenotype is that the defect in meiotic recombination worsens with age. This implies that the DSB-1 protein retains some residual DSB-promoting activity in the absence of its paralog, but also indicates that the requirement for DSB-2 becomes more acute in older germ cells. Interestingly, CO distribution has also been found to differ between young and old WT C. elegans oocytes C. elegans reproductive system has substantial plasticity in this regard, as the duration of progression through meiotic prophase varies markedly with both sex and age and can be modulated dramatically in the female germ line by the availability of sperm An intriguing aspect of the Strains were maintained at 20°C under standard conditions. Experiments were performed at 20°C unless otherwise noted. Strains used in this study:unc-119 III; ruIs32  III mnT12 AV334 unc-119 III; ruIs32  IIIAZ212 dsb-2(me96) II 4X outcrossedAV477 rol-1(e91) dsb-2(me96) IIAV501 rol-1(e91) dsb-2(me96) unc-52(e998) IIAV511 rol-1(e91) dsb-2(me96)/mnC1 [dpy-10(e128) unc-52(e444)] IIAV539 meIs8[unc-119(+) pie-1promoter::gfp::cosa-1] II ;; ItIs37[unc-119(+)pie-1::mcherry::histoneH2B]; ltIs38AV727 dsb-2(me97) meIs8[unc-119(+) pie-1promoter::gfp::cosa-1] II ;; ItIs37[unc-119(+)pie-1::mcherry::histoneH2B]; ltIs38AV758 meIs8[unc-119(+) pie-1promoter::gfp::cosa-1] IIAV630 spo-11(ok79)/nT1 IV; +/nT1[qIs51] VAV645 chk-2(me64) rol-9(sc148)/unc-57(e369) rol-9(sc148) VAV146 chk-2(me64) rol-9(sc148)/sC4(s2172)[dpy-21(e428)] VAV660 +/nT1 IV; sun-1(gk199)/nT1 VVC292 +/nT1 IV, him-17(ok424)/nT1 VVC255 +/nT1 IV; rad-50(ok197)/nT1 [unc-?(n754) let-? qIs50] VAV158 dpy-13(e184) rad-51(lg8701) IV/nT1[let-?(m435)] TG9 rad-54 and tag-157(ok615) I/hT2[gli-4(e937) let(9782) qIs48] I; IIIVC531 zhp-3(me95)/hT2 [bli-4(e937) let-? (q782) qIs48] IAV449 msh-5(me23)/nT1 IV; +/nT1[qIs51] VAV603 cosa-1(tm3298)/qC1[qIs26] IIIAV596 +/nT1 IV; syp-1(me17)/nT1 VAV307 htp-1(gk174) IV/nT1[unc-?(n754) let-? qIs50] AV393 htp-3(y428) ccIs4251 I/hT2[bli-4(e937)let-?(q782) qIs48] .TY4986 +/nT1 IV; rad-50(ok197)/nT1[qIs51] VAV473 htp-1(gk174)/nT1[ unc-?(n754) let-? qIs50] IV; rad-50 (ok197)/nT1 [qIs51] VAV443 Bristol (N2) wild typeCB4856 Hawaiian wild typedsb-2(me96) allele was isolated in a genetic screen for meiotic mutants exhibiting defects in chiasma formation or chromosome organization in diakinesis-stage oocytes, conducted in collaboration with M. Hayashi me95 and me96, were identified based on the presence of univalents at diakinesis in a the subset of F2s (from independent F1s) and were recovered by plating of siblings; repeated outcrossing (with N2) and selecting for the mutant phenotype removed the chromosome fusion as well as the GFP::H2B transgene. Mapping, complementation testing and sequencing revealed that me95 is an allele of zhp-3, containing a C-to-T transition that results in a premature stop at codon 348 of the predicted 387 amino acid coding sequence of K02B12.8a. Mapping of the me96 mutation (below) indicated that it identified a new component of the meiotic machinery.The me96 near genetic map position 20 on the right side of chromosome II. To select for informative COs near this region, rol-1 me96 unc-52 worms were crossed with CB4856 males, and Rol non-Unc F2 progeny were selected and genotyped for SNP pkP2117 at genetic map position 17.9 to select for COs occurring between this marker and unc-52 (genetic map position 23). Informative recombinants were assessed for me96 phenotype and typed using additional SNP markers in the region, narrowing down the position of me96 to a 165 kb interval (between SNP markers uCE2-2315 and uCE2-2332) comprising 36 candidate genes. RNAi of candidate genes was performed using bacterial clones from the RNAi feeding library F26H11.6  phenocopied the me96 mutation, eliciting a mixture of bivalents and univalents at diakinesis. Sequencing identified a T-to-A transversion generating an early stop at codon 14 (TTA = >TAA) of the predicted F26H11.6 coding sequence  in the me96 mutant.Initial SNP mapping based on the methods of dsb-2 allele (me97) was isolated independently in a screen for mutants with altered numbers of GFP::COSA-1 foci (which mark CO sites in late pachytene); me97 fails to complement me96 and contains a premature stop at codon 168, further confirming the identity of F26H11.6 as the dsb-2 gene. Except where otherwise noted, all analyses were conducted using the me96 allele.A second dsb-2(me96); rol-1(e91) hermaphrodites (where rol-1 is a marker with no meiotic defects) were counted. The number of eggs counted for each day is: 573 (day 1), 879 (day 2) and 488 (day 3), with an average of 243 eggs per hermaphrodite over the three day interval.L4 hermaphrodites were picked to individual plates, allowed to lay eggs, and transferred to fresh plates every 24 hours for three days. Hermaphrodites start laying eggs after they transition from L4 to adult, and lay most eggs in the first three days. Inviable embryos that do not hatch are indicative of autosomal mis-segregation, while male progeny indicate X-chromosome mis-segregation. Eggs from eight dsb-2 worms, 1 day and 2 day post L4 respectively; 43 and 57 for WT worms, 1 day and 2 day post L4 respectively.Numbers of DNA bodies present in diakinesis oocytes were assessed in intact adult hermaphrodites of indicated age, fixed in ethanol and stained with 4′,6-diamidino-2-phenylindole (DAPI) as in dsb-2 24 h, n = 127; dsb-2 48 h, n = 101; WT 24 h, n = 76; WT 48 h, n = 78. Numbers of GFP::COSA-1 foci in late pachytene nuclei of live anesthetized worms (0.1% tricaine and 0.01% tetramisole in M9 buffer ) were quantified by taking 3D image stacks on a DeltaVision microscope. GFP foci were counted in the last five rows of pachytene nuclei; only nuclei completely contained within the stack were scored, and nuclei with features indicative of apoptosis  were excluded. 24 h control data were from fixed immunofluorescence images Immunofluorescence was conducted as in The following primary antibodies were used at the indicated dilutions in PBST with 0.5% BSA: guinea pig anti-HIM-8 (1∶500) dsb-2 mutant gonads  using the C-terminal 100 amino acids of F26H11.6 as the immunogen. Specificity of the antibody was demonstrated both by the lack of chromatin staining in immunofluorescence analysis of t gonads  and by Wt gonads .spo-11(me44) gonads.Rat anti-RAD-51 antibody was generated using a His-tagged fusion protein expressed from plasmid pET28a containing the entire RAD-51S coding sequence All secondary antibodies were Alexa Fluor goat from Invitrogen used at 1∶200 dilution in PBST with 0.5% BSA.Immunofluorescence images were acquired using the DeltaVision microscopy system (Applied Precision) and deconvolved using softWoRx software. Images shown are maximum-intensity projections of Z-stacks acquired at 0.3 µm intervals.dsb-2(me96) mutant germ lines (in which the abundance of RAD-51 foci was low throughout). Quantitation was carried out on deconvolved 3D image stacks using SoftWoRx software; only nuclei that were completely contained with in the image stack were scored. Occasional atypical nuclei with condensed, bright DAPI signals were excluded. Numbers of nuclei scored: WT, n = 335; dsb-2, n = 196.For each wild-type germ line evaluated, RAD-51 foci were quantified in 8 contiguous rows of pachytene nuclei from the region where foci were most abundant. The average distance (in rows of nuclei) between the position of this peak and the end of the transition zone was calculated for wild-type germ lines, and this distance was used to define the corresponding regions to be scored in For each genotype, sixty adult worms (24 hours post-L4) were picked into M9+0.05% Tween 20, washed gently three times, then lysed by resuspension in 2× Laemmli Sample Buffer (Bio-Rad). Gel electrophoresis was performed on a 4–15% Criteriot TGX gradient gel (Bio-Rad), followed by transfer of proteins to a PVDF membrane. Blots were probed with rabbit anti-DSB-2 (1∶1000 in 5% milk) for 2 hours, followed by HRP-conjugated secondary antibody and detection by ECL.spo-11(ok79) mutant dsb-2 and age-matched spo-11 mutants. The dsb-2 worms also carried the rol-1 marker, which does not affect meiosis. This assay tends to underestimate the incidence of achiasmate chromosomes, as some lie too close together to be resolved. Numbers of nuclei scored were: 71 and 76 for dsb-2 worms, untreated and irradiated respectively; 76 and 45 for spo-11 worms, untreated and irradiated respectively.Worms were exposed to 1 kRad (10Gy) of gamma-irradiation using a Cs-137 source. The 1 kRad dose was chosen based on its sufficiency to restore chiasmata to 95% of chromosome pairs in affected nuclei of the rad-50 and htp-1; rad-50 mutants were irradiated at 24 hours post-L4, and stained with DAPI, RAD-51 antibody and DSB-2 antibody. Germ lines from dsb-2 mutants were irradiated at 48 hours post-L4 and stained with DAPI and RAD-51 antibody.Worms were exposed to 5 kRad (50Gy) of gamma-irradiation using a Cs-137 source. Formation of RAD-51 foci was assessed by immunofluorescence in gonads dissected and fixed 1 hour after irradiation. Germ lines from Figure S1Multiple sequence alignment of DSB-2 family proteins. Multiple sequence alignment of DSB-2 family proteins produced by T-Coffee, a consistency-based aligner  (TIF)Click here for additional data file.Figure S2Phylogenetic tree of DSB-2 family proteins. Average distance phylogenetic tree of DSB-2 family proteins generated using BLOSUM62 matrix. This tree is based on the T-Coffee alignment. This protein family is highly divergent, with the two closest members showing only 51% identity (CBG03330_briggsae and RP45881_remanei). The F26H11.6 (DSB-2) protein shows <30% identity with any member of the family. Multiple sequence alignment and associated phylogenetic trees suggest that an early duplication event occurred before the species separated, and that the F26H11.6 branch is diverging more rapidly. The slightly more conserved F08G5.1 (DSB-1) branch reflects the evolutionary relationship of the species (TIF)Click here for additional data file.


In this review, we summarize the current knowledge about the molecular and functional interactions between polycystins and multiple components of the cellular Ca2+-signaling machinery. In addition, we discuss the relevant downstream responses of the changed Ca2+ signaling that ultimately lead to increased proliferation and increased apoptosis as observed in many cystic cell types.The cystic phenotype in autosomal dominant polycystic kidney disease is characterized by a profound dysfunction of many cellular signaling patterns, ultimately leading to an increase in both cell proliferation and apoptotic cell death. Disturbance of normal cellular Ca PKD1 or PKD2 genes, which encode the proteins polycystin-1 and -2, respectively. Mutations in the PKD1 gene account for approximately 85 % (ADPKD type 1), and mutations in the PKD2 gene account for approximately 15 % (ADPKD type 2) of the affected individuals  lowered the open probability  upon agonist stimulation [2+ levels and its activity may be tightly controlled. Among the other polycystin-2-interacting proteins, polycystin-1 [An interaction between the C-termini of polycystin-1 and polycystin-2 is considered to be important for activation of the Caactivity , 21. Thiactivity , 81. Themulation . A simil elegans . The opebability . The obs812 site . Polycysthe IP3R , 58. Moreraction  tion [3R . Disrupth effect . It is cresponse . At variresponse . The IP3 channel . The app channel , 38, 58,epletion , can posepletion . Syntaximulation . Polycyscystin-1 , α-actincystin-1 , mammalicystin-1 , and fibcystin-1  have bee2+-release properties. This altered RyR2 function could play a role in the development of cardiovascular abnormalities in ADPKD patients [Polycystin-2 was also found to bind and regulate the RyR2 in the sarcoplasmic reticulum (SR) of cardiac myocytes . The C-tpatients .3R thereby inhibiting IICR [3R ligand-binding domain similarly to the binding site for polycystin-2 but with an opposite effect. It was therefore proposed that polycystin-1 and polycystin-2 both interact with the IP3R in a complementary way to maintain a balance of proper ER-mediated Ca2+ signaling [2+ transients. These data were interpreted as due to an inhibition of the Ca2+ leak across the ER membrane. These studies clearly illustrate the importance of polycystins in maintaining proper ER Ca2+ homeostasis [2+ responses, was the identification of a P100 C-terminal fragment of polycystin-1 that is localized in the ER and interacts with STIM1 [2+ currents. This would then lead to decreased store filling and subsequently to diminished agonist-induced Ca2+ transients [2+ concentration and store-operated Ca2+ entry (SOCE) [2+ signaling. Mechanistically, polycystin-1 was proposed to increase the interaction between the IP3R and STIM1, which thereby inhibited Ca2+ release and SOCE [3R/STIM1. It should be pointed out, however, that in a study comparing STIM1−/− and wild-type MEF cells, no evidence was found for a direct interaction between STIM1 and the IP3R, but the effects on IICR were attributed to changes in the connections between the ER and plasma membrane [On the one hand, polycystin-2 can act as a regulator of other ER/SR channels, but on the other hand, it has its own channel properties that may be controlled by different cellular parameters. An obvious candidate for the regulation of polycystin-2′s channel properties in the ER is polycystin-1. As stated above, models were proposed for interaction of polycystin-1 in the plasma membrane with polycystin-2 in the ER , 81. Alting IICR . The intignaling . A diffeignaling , showingeostasis . Anotherth STIM1 . P100 pransients . It shouy (SOCE) , 95. Thiy (SOCE) . The natand SOCE  membrane .2+ release were mostly obtained by exogenous expression of polycystin-1 or its C-terminal fragments. The properties of endogenous polycystin-1 as obtained from knock-down experiments are much less documented. In studies of polycystin-1 haploinsufficiency in renal cells [2+ concentration in the polycystin-1+/− as compared to the wild-type cells. Moreover, vascular smooth-muscle cells from polycystin-1+/− mice exhibited a decreased agonist-induced Ca2+ release as compared to the wild type [2+-channel properties. Disturbance of this complex by either polycystin-2 or polycystin-1 knock-out is then expected to result in a decreased Ca2+-release activity. This is exactly what was recently found for the effect on IICR in a model system of plasma membrane-permeabilized cells upon lentiviral knock-down of either or both polycystins [2+ release from the ER and knock-down of either polycystin decreased the apparent sensitivity of IICR [2+ fluxes. An interesting view that was presented in a model by the Guggino group [2+ fluxes and promoting a situation with more filled stores and inactivated SOCE with Ca2+ influx via plasma membrane (or cilia)-localized polycystin-2  and/or Ca2+-dependent phosphodiesterase (PDE) [2+ and cAMP is, however, very complex, as there are nine different genes encoding transmembrane-domain ACs (AC1–9) along with numerous splice variants of the gene encoding soluble ACs, while also the cyclic nucleotide-PDE superfamily consists of over 30 genes [2+, either directly or via calmodulin. A common theme in the regulation of cAMP production is the pronounced intracellular compartmentalization. Three modes of cAMP microdomain/compartment formation are found: via (a) lipid rafts, (b) A-kinase anchor proteins (AKAPs), and (c) targeting of soluble AC to cellular organelles [2+ that inhibits the Ca2+-sensitive AC5 and AC6. Malfunction of polycystin-2 as a Ca2+ channel may reduce the local Ca2+ concentration in the cilium and thereby activate AC5/6. Other mechanisms like mutations of the transcription factor hepatocyte nuclear factor-1ß (HNF-1ß) were found to inhibit the expression of PDE4C and thereby increase cAMP levels [Pkd2 itself [2+, which in ADPKD would result in decreased PDE activity and a higher cAMP concentration [2+-store depletion and STIM1 translocation [2+ concentration in the ER led to recruitment of ACs through a process involving STIM1 [Disturbed Capathways . Among tpathways , possiblpathways . The reapathways , 104. A se (PDE) . The int30 genes . Moreoveganelles . It can ganelles . A proteP levels . As HNF-2 itself , downreg2 itself . PDE1 is2 itself . Importantration . An addilocation . These dng STIM1 . This meng STIM1 . In viewng STIM1 , 96.2+ concentration [2+ restriction was associated with an elevation in B-Raf protein levels. In these conditions, cAMP stimulates B-Raf/MEK/ERK signaling in a sarcoma (Src)- and Ras-dependent manner. Moreover, the activity of AKT, a negative regulator of B-Raf, was decreased by Ca2+ restriction. Inhibition of AKT or PI3-K also allowed cAMP-dependent activation of B-Raf and ERK at normal Ca2+ levels. These results suggest that Ca2+ restriction causes an inhibition of the PI3-K/AKT pathway, which relieves the inhibition of B-Raf to allow the cAMP growth-stimulated phenotypic switch [2+ concentration was found to be 20 nM lower in cyst-derived ADPKD cells compared with normal cells. Elevation of the cytosolic Ca2+ concentration in ADPKD cells increased AKT activity and blocked cAMP-dependent B-Raf and ERK activation. Thus, an increase in the cytosolic Ca2+ concentration was able to restore the normal anti-mitogenic response to cAMP [2+-channel inhibition by verapamil accelerated polycystic kidney-disease progression [2+ concentration. The differential effects of polycystin-1 expression on the cytosolic Ca2+ concentration and downstream effects could reflect the use of different cell types such as immortalized cells versus primary cells [In general, cystic epithelia in ADPKD have high levels of cAMP and also of mitogen-activated protein kinase (MAPK) activity. This signaling pathway, involving cAMP-dependent PKA, stimulates the proliferation of cells from ADPKD cysts, but not cells from normal human kidney –113. In ntration , 115. Cac switch , 114. Thgression . On the gression  or expregression , 118 resry cells .Fig. 2SiTsc2, in a complex with hamartin, the product of Tsc1, is the primary negative regulator of mTORC1. Tuberin is a GTPase-activating protein (GAP), which regulates Rheb, a small G-protein belonging to the Ras superfamily. Hamartin and tuberin form a heterodimer, which converts Rheb-GTP to the inactive Rheb-GDP. Rheb is an activator of mTORC1 and conversion of Rheb to the GDP-bond form inactivates the mTORC1 pathway [Pkd1 gene in conditional knock-out mice [2+  [2+ concentration in cystic cells and mTORC1 via CaMKKβ-dependent regulation of AMPK is, however, not yet established. A relation between AMPK activity and cyst formation was, however, found by using metformin, a drug in wide clinical use as a pharmacological activator of AMPK [Cyst-lining epithelial cells exhibit higher levels of mTOR signaling as compared to normal epithelial cells . mTOR is pathway –123. The pathway  or indir pathway . A promiout mice . The regout mice . Among tignaling , 115. Anignaling . LKB1 acs or AKT . Next to(CaMKKβ) . The lin of AMPK . Stimula of AMPK .2+-dependent way. Wnt signaling, which was shown to be regulated by polycystin-1 [2+ pathway influences both canonical and non-canonical pathways [2+ influx can switch off the canonical and activate the non-canonical pathway [2+ signaling may thus both contribute to a GSK3-dependent increase in mTOR activity [Additional pathways may converge on mTOR signaling in a Cacystin-1 , can actpathways . Inversipathways . It can  pathway . Over-ac pathway . This is pathway , 134–136 pathway  Fig. 2)2+-depend pathway . Moreove pathway . In ADPKactivity .0/G1. This process requires polycystin-2 as an essential cofactor [−/− mouse resulted in polycystic kidney disease with concomitant down-regulation of anti-apoptotic Bcl-2-family proteins and massive apoptotic cell death [Kidneys from ADPKD patients have high levels of apoptosis in addition to increased cellular proliferation –141. Thicofactor . A decrecofactor . Roscovicofactor , 148. Recofactor . Tubularcofactor , 151 andcofactor , as wellcofactor –141. Miccofactor , 154. Whcofactor , this wacofactor . Similarll death . A direcll death . A markell death , 160, anll death . In the ll death . Overexpll death , 162–1652+ signaling is implicated in the phenotypic feature of ADPKD cells showing elevated rates of both proliferation and apoptosis, but the downstream mechanisms are not fully resolved. It was found that polycystin-1 induces resistance to apoptosis and normal tubulogenesis through the PI3-K/AKT pathway [2+ concentration, down-regulation of PI3-K/AKT and activation of B-Raf/ERK in ADPKD cysts [2+ signaling could be a primary event in ADPKD and may be responsible for the switch to a proliferative phenotype with an elevation in B-Raf protein levels and cAMP-stimulated, Ras-dependent activation of B-Raf and ERK [PKD2 transgenic mouse model resulted in renal-cys formation via B-Raf signaling, probably by acting as a dominant negative modulator for polycystin function and downstream Ca2+ signals [Pkd2+/− vascular smooth-muscle cells also have an abnormal phenotype with a defective Ca2+ signaling with decreased levels of the cytosolic Ca2+ and an increased cellular cAMP concentration, which is probably underpinning increased proliferation and apoptosis [2+ concentration apparently provoked increased proliferation in these vascular smooth-muscle cells but the effect in this cell type was independent of cAMP/B-Raf signaling [Ca pathway  Fig. 3)2+ signalKD cysts . As alre and ERK , 115. Al signals . Pkd2+/poptosis . The lowignaling .2+ signaling in the form of activation of Ca2+ oscillations [2+ concentration which led to activation of cell proliferation. The proposed mechanism involved non-capacitative Ca2+ entry (NCCE), which was proposed to be negatively regulated by polycystin-1. The loss of polycystin-1 resulted in NCCE activation and increased Ca2+ oscillations. The downstream effects were related to activation of the calcineurin/nuclear factor of activated T cells (NFAT) pathway. The explanation could be that Ca2+ oscillations rather than the basal Ca2+ concentration are crucial for activation of the NFAT-dependent cell proliferation [A divergent mechanism was proposed, in which disturbed polycystin-1 function affected cell proliferation by an increase in intracellular Callations  Fig. 2)2+ signalferation .2+ content by polycystins [2+ content is determined by the activity of Ca2+ pumps and by the Ca2+-leak rate via different pathways. The resulting ER Ca2+ load is a primary determinant of the extent of ER-to-mitochondrial Ca2+ transfer and pro-apoptotic Ca2+ signaling [2+-leak pathway and in this way control the degree of ER Ca2+ filling [2+ release from the ER in response to apoptotic stimuli, and conversely, its loss in ADPKD would lead to an increased apoptosis [2+ are probably tightly regulated by many other cellular factors and interacting proteins. Not only is polycystin-2 a Ca2+ channel but it also interacts with the two main intracellular Ca2+-release channels, IP3Rs and RyRs, and with various plasma-membrane TRP channels. Polycystin-1 controls the activity of polycystin-2 directly and also in an indirect way via PI3-K/AKT signaling. Decreased PI3-K/AKT signaling in ADPKD would thereby lead to a profound remodeling with increased IICR and SOCE 2+ contenignaling . Polycys filling . Normal poptosis . Althougrylation . Increasrylation , 170.2+ signaling is clearly a very early event in the development of the cystic phenotype of renal cells. This phenotype is characterized by a concomitant activation of both cell proliferation and apoptotic cell death. Despite the increased proliferation as an invariable component of cystogenesis, there is seldom progression towards renal carcinoma [2+ signaling by direct or indirect interaction with many proteins of the cellular Ca2+ toolkit. It becomes increasingly evident that polycystin dysfunctions lead to a profound remodeling of cellular Ca2+ signaling and provoke changes in the spatio-temporal modes of Ca2+ signaling. This could lead to changes in the occurrence of different Ca2+-signaling microdomains located at the cilia or at the ER-mitochondria junctions, and it could lead to oscillatory Ca2+ signals that may evoke nuclear responses. It will be a challenge for future research to experimentally detect the subcellular Ca2+-signaling microdomains and to identify their downstream responses that result in the increased proliferation and increased apoptosis responses, which are characteristic for the cystic phenotype.In conclusion, disturbed or remodeled cellular Caarcinoma . Overexparcinoma . ADPKD earcinoma , 172. Bo