Membranous nephropathy (MN), the leading cause of nephrotic syndrome in adults, is characterized by the deposition of subepithelial immune deposits. Most of the cases are primary, while only approximately 25% of the cases are secondary to some known diseases. Recently, MN has been considered to be a possible presentation of chronic graft-versus-host disease (GVHD) of the kidney in allogeneic hematopoietic stem cell transplantation (HSCT) patients. In autologous HSCT populations, there have been scarce reports of associated MN, as a result of immune dysregulation leading to systemic autoimmunity and miming chronic GVHD.We report an exceptional case of MN associated to an acute renal failure occurring within days following an autologous HSCT indicated by multiple myeloma. There was no evidence of GVHD or myeloma relapse. A complete remission of nephrotic syndrome with normalization of renal function were rapidly obtained by corticosteroid therapy.This is the first published case of acute renal failure due to MN occurring in the acute phase of an autologous HSCT. These findings support the antibodymediated autoimmune glomerular disease. Acute and even chronic renal injury are known complications of the Hematopoietc Stem Cell Transplant (HSCT) , 2. TheyGlomerulopathies represent only a small proportion of such injury occuring in 1 to 6% of allogenic HSCT recipients –6. They With the emergence of autologous HSCT, GVHD should not exist. Nevertheless, Autologous GVHD has been described in up to 10% of patients after autologous HSCT , with inRecently, there have been scarce reports of associated MN in autologous HSCT population . Some au3, hemoglobin 9.7 g/dl, platelet count 226,000/mm3 and prothrombin time 32 s (79%). Erythrocyte sedimentation rate was 139/145,serum total protein was 7, 8 g/dl , Immunophenotyping: peak monoclonal IgG Kappa, albumin 3,8 g/dl, creatinine 78 μmol/l, blood urea nitrogen 7 mmol/l, calcemia 2,2 mmol/l, blood uric acid 532 μmol/l. The 24-h urinary excretion of total protein was 1 g/d with Bence-Jones proteinuria of 0, 6 g. Bone marrow aspiration showed myeloma cell infiltration in the bone marrow (31% of dystrophic plasma cells). The diagnosis of IgG Kappa multiple myeloma was made and he began, in December 2013, his induction treatment with Thalidomide-Dexamethasone receiving 3 cycles with good response. Then, he underwent in June 2014 autologous SCT with high-dose melphalan.A 54-year-old male with history of hypertension for 2 years was investigated for normochromic normocytic anemia in September 2013. Initial Laboratory data were: leukocyte count 6700/mmDuring the second week, Autograft was complicated by a urinary tract infection and sepsis Staphylococcus hemolyticus with development of acute renal failure, the blood creatinine level reaching 336 μmol/l. Renal function improved on antibiotics but has not normalized so the patient was referred to Nephrology when his blood count was normalized for further evaluation of his renal failure. At Nephrology (day 29), the blood creatinine level was 260 μmol/l and multiple myeloma was in total remission. However, there was a progressive increase in proteinuria becoming nephrotic. Bence- Jones proteinuria remained negative. A kidney biopsy was then performed showing features of membranous nephropathy Fig. . No secoThe patient was put under renin-angiotensin system blockers. Renal function improved (creatinine 160 μmol / l after 3 months), the proteinuria remained oscillating between 3,5 and 11 g / l. After one year of monitoring, diagnosis was discussed again on the basis of a recent bibliography and a corticoid treatment, prednisone at a dose of 1 mg / kg / day was introduced targeting an autoimmune process. The outcome was favorable with a decrease of proteinuria remaining between 0.6 and 0.8 g / d and a creatinine down to 103 μmol/l at 3 months of treatment. Corticosteroid degression was started after 4 months motivated by the good evolution and development of cortico-induced diabetes. Total duration of corticotherapy was 8 months. At last check , renal function was stable (creatinine at 102 μmol/l), PLA2R autoantibodies and proteinuria were negative.GVHD is a potentially lethal condition in which transplanted donor leukocytes (graft) attack the recipient’s tissue (host) . GVHD ofPost-allogeneic HSCT glomerulopathies most often manifest as nephrotic syndrome caused primarily by MN . GVHD isWith the emergence of autologous HSCT, GVHD and its complications, quite common in allogeneic transplantation,were expected to be eliminated. However, autologous GVHD has been described in some patients after autologous hematopoietic HSCT.Recent studies have indicated that two major factors are necessary for the induction of autologous GVHD. The first factor was a disruption of thymic-dependent immune reconstitution and the second one was a failure to re-establish peripheral self-tolerance .Once the alloreactive T cells are released to the periphery, they can be eliminated using a T-cell-dependent regulatory system, however, due to the lymphoablative preparative regimen that HSCT patients undergo, this system is not functional , 16.On another hand, autologous GVHD has been thought of as an autoimmune syndrome and a milder form of GVHD than its counterpart in allogeneic transplantation. In recent studies, the presence of auto-antibodies in relation to the HSCT provides evidence for the involvement of the B cell. A possible theory on how B cells can contribute to chronic GVHD is that the reconstituted B cells after myeloablative conditioning may have impaired immune tolerance of peripheral B cells, leading to the production of autoantibody in chronic GVHD .In autologous HSCT populations, there have been reports of associated glomerular disease, as a result of any type of immune dysregulation. Based on literature, 6% of all patients with glomerulonephritis after HCT had received Autologous transplants , 18, 19.MN is encountered very rarely following autologous HSCT (3% postautologous vs 97% postallogeneic HSCT [Nephrotic syndrome was described to be a late event after HSCT either post alloneneic HSCT or autologous HSCT. Indeed, nephrotic syndrome occurring in allogeneic HSCT patients has been considered to be a possible presentation of chronic GVHD of the kidney and a decrease in immunosuppressive medication after HSCT was associated with the occurrence of nephrotic syndrome within 9 months in 63% of patients , 20. In Here we presented a case of a MN diagnosed few days after a HSCT without features of acute GVHD. Primary MN was initially evoked, however the absence of anti-PLA2R antibody rendered the possibility of the coincidental development of primary MN less likely. More, our patient showed complete remission to steroid treatment which is in favor of post-HSCT MN. Indeed, as opposed to primary MN, corticosteroids, usually are the first line of treatment for post-HSCT MN , 14, 21.Although post HSCT MN likely represents a late manifestation of chronic GVHD in allogeneic HSCT, it may also occur after autologous HSCT. Auto immunity seems to be on the basis of its occurrence as supported by the glomerular deposition of IgG and complement components. Auto immunity could be acute and involve only the kidneys as seen in our case. Corticosteroids are the principal treatment toil and may be sufficient.Through this observation, we recommend more screening protocols of proteinuria and renal function post HSCT.


This paper elucidated unconventional mechanism of 4-aminopyridine-stimulated glutamate release from neurons and non-neuronal cells which proceeds in the absence of external Ca2+. In brain nerve terminals, primary neurons and platelets 4-aminopyridine induced the exocytotic release of glutamate that was independent of external Ca2+ and was triggered by the sequestration of Ca2+ from intracellular stores. The initial level of 4-aminopyridine-stimulated glutamate release from neurons in the absence or presence of external Ca2+ was subequal and the difference was predominantly associated with subsequent tonic release of glutamate in Ca2+-supplemented medium. The increase in [Ca2+]i and the secretion of glutamate stimulated by 4-aminopyridine in Ca2+-free conditions have resulted from Ca2+ efflux from endoplasmic reticulum and were abolished by intracellular free Ca2+ chelator BAPTA. This suggests that Ca2+ sequestration plays a profound role in the 4-aminopyridine-mediated stimulation of excitable and non-excitable cells. 4-Aminopyridine combines the properties of depolarizing agent with the ability to sequester intracellular Ca2+. The study unmasks additional mechanism of action of 4-aminopyridine, an active substance of drugs for treatment of multiple sclerosis and conditions related to reduced Ca2+ efflux from intracellular stores.4-aminopyridine is commonly used to stimulate neurotransmitter release resulting from sustained plasma membrane depolarization and Ca This in turn can lead to incorrect interpretation of the results in experimental research and become a likely cause of unpredictable side-effects for medical practice. This study has been focused on the action of 4-aminopyridine (4-AP), well known blocker of voltage-activated K2+ influx through potential-sensitive Ca2+-channels12+. The stimulation of gamma-aminobutyric acid (GABA) and acetylcholine release was dependent on the presence of external Ca2+, whereas no clear Ca2+-dependence was observed for glutamate release2+-channels2-adrenoreceptorsBesides the prevention of presynaptic membrane repolarization and increasing the propagation of action potential along axons, 4-AP can promote Ca2+. The aim of presented study was to study the origin and mechanism of glutamate release stimulated by 4-AP in excitable and non-excitable cells in the absence of external Ca2+. The comparative analysis of the data obtained revealed new properties of 4-AP action common for all types of cells tested.This supports the view that the mode by which 4-AP stimulates secretion in different cell types is more complex and, yet, not fully uncovered. In light of the diverse mechanisms of 4-AP action further research is required to discriminate between neurotransmitter release stimulated in the absence and presence of external Cath day of culturing  increased by 6% compared to Ca2+-free conditions. The same difference was inherent for subsequent release of the dye: nerve terminals released 64.8 ± 7.4% and 69.7 ± 6.7% of accumulated dye in the absence of Ca2+ and in the presence of 2 mM Ca2+, respectively. This property of 4-AP was also demonstrated in the experiments with neurons: Ca2+-dependent release was 12% higher than Ca2+-independent. The difference in steady-state level of dye uptake in 2+.The next step was to compare the 4-AP-stimulated secretion of synaptic vesicle constituents in the absence and presence of external Caependent . The accTo exclude the unspecific effects of 4-AP due to its base properties or possible interaction with the dye additional experiments were carried out for the acidification and neurotransmitter storage in isolated synaptic vesicles. Synaptic vesicles were loaded with the pH-sensitive dye acridine orange that tends to form dimers and multimers after protonation at acidic pH. This led to the decrease in green fluorescence that is inherent for monomeric dye.L-glutamate induced additional acidification of synaptic vesicles , which iNeurons and platelets have cytosolic and secretable pools of glutamate. The former depends on the functioning of high-affinity glutamate transporters  on plasma membrane, vesicular glutamate transport and the activity of enzymes that metabolize glutamate. The secretable pool of glutamate represented by synaptic vesicles in neurons and dense secretory granules in platelets is replenished by vesicular glutamate transporters VGLUT1/289To estimate the release of endogenous glutamate that follows 4-AP stimulation the glutamate dehydrogenase assay was used. For quantitative analysis the activity of glutamate dehydrogenase was calibrated with the different concentrations of exogenous glutamate at the end of experiments.2+-free medium: after application of 250 μM 4-AP platelets released 3.45 ± 0.5 nmol of L-glutamate per mg of total platelet protein. The initial level of 4-AP-stimulated glutamate release from neurons and isolated brain nerve terminals in the absence or presence of Ca2+ was subequal, and the difference was predominantly associated with the following tonic release of glutamate in Ca2+-supplemented medium. In isolated brain nerve terminals and primary neurons in Ca2+-free medium 250 μM 4-AP induced the release of 16.2 ± 1.8 nmol and 25.5 ± 3.4 nmol of endogenous glutamate per mg of total synaptosomal/neuronal protein (at 5th min), respectively  enhanced the surface exposure of CD63  of Fluo-3 and FURA-2 after in situ calibration consisted of 2,700 nM and ~425 nM, respectively, which differed significantly from Kd determined in the solution. 4-AP induced transient (but with rising 4-AP concentration constant) increase of [Ca2+]i in platelets from basal 40.2 ± 8.3 nM to 185.5 ± 16.1 nM 0 (%), where F0 was the level of fluorescence before cell stimulation and dF was the change in fluorescence intensity after the application of 4-AP. The monitoring of [Ca2+] changes in Mag-Fluo-4-loaded platelets and neurons allowed to demonstrate that 4-AP (250 μM) provoked the sequestration of Ca2+ from endoplasmic reticulum (ER) in the absence of external Ca2+: the level of Mag-Fluo-4 fluorescence intensity decreased by 23% and 28.5% from initial level in neurons and platelets, correspondingly  that was abolished by uncoupling agent carbonyl cyanide m-chlorophenyl hydrazone (CCCP).There were no detectable changes of mitochondrial i and endogenous glutamate release were observed regardless of the presence of Ca2+ in the medium. The external Ca2+-independent equal/subequal release of synaptic vesicle constituents  4-AP was unable to stimulate the release of AO and endogenous glutamate from isolated synaptic vesicles , indicat2+ 4-AP-stimulated glutamate release was accompanied by the destaining of FM1-43-loaded synaptic vesicles , their size (~150 nm diameter), the measured platelet count per mg of protein and the established release of endogenous glutamate from platelets during the stimulation  the conc2+]i rise in neurons and platelets in the absence of extracellular Ca2+. The absolute [Ca2+]i measured with Ca2+ sensitive probes 232+ signalling depends on the proximity of the Ca2+ sensor and the Ca2+ channel. The Ca2+ influx through a single Ca2+ channel (nanodomain) requires the location of the Ca2+ sensor in close proximity to the channel (within 50 nm) for efficient signalling, whereas the summation of Ca2+ influxes from clusters of Ca2+ channels (microdomain) can trigger exocytosis in active zones within 200 nmThe most important finding is the ability of 4-AP to stimulated [Cae probes  increase]i in nM . However2+ chelator, BAPTA, capable of rapid binding with Ca2+ and effective buffering of all sorts of Ca2+ signals was applied to confirm the direct coupling between the 4-AP-induced Ca2+ sequestration and endogenous glutamate release (2+ (2 mM EGTA) the accumulation of AO in BAPTA-loaded cells was lower  4-AP stimulates 1–the influx of Ca2+ stores. This enables fast cooperation between the initial stimulus, the generation of second messengers and the release reactions at growing plasmalemmal surface during the pseudopodia formation. The plasma membrane and ER dimension, inherent for neurons and platelets, is essential for cooperation in regulation of the Ca2+-dependent processes within the microdomainsPlatelet plasma membrane forms multiple invaginations into the cytoplasm and creates a network of the channels . This network inside of platelet is in vicinity of the dense tubular system 2+ via membrane potential-driven uptake. Agonist-stimulated elevations of [Ca2+]i evoke rapid and transient increase of mitochondrial [Ca2+], which can be prevented by pretreatment with a mitochondrial uncoupler2+-sensitive probes targeted to ER (Mag-Fluo-4) or mitochondria (Rhod-2) enabled the determination of the Ca2+ store responsible for 4-AP-stimulated Ca2+ efflux. In neurons and platelets 4-AP caused the decrease of [Ca2+] in ER similarly to that induced by thapsigargin application , minimum essential medium, FBS, poly-D-lysine, A23187 and digitonin were purchased from Sigma-Aldrich (USA), Ficoll-400–from Amersham (UK). Acridine orange, rhodamine 6G, FM1-43, Fluo-3/AM, FURA-2/AM, Mag-Fluo-4/AM and Rhod-2/AM were obtained from Molecular Probes (USA). All chemicals used were of analytical grade. The water solutions were made with deionised water.L-glutamic acid, 4-aminopyridine, glutamate dehydrogenase , NaCl, KC1, MgClAnimal care, blood sampling and euthanasia, were carried out in accordance with the European Guidelines and International Laws and Policies and were approved by the Institutional Animal Care and Use Committee, Palladin Institute of Biochemistry, NAS of Ukraine (protocol N2 from 12.04.2014).2+ concentrations were set with Ca2+/EGTA buffers.The male rats were sacrificed by decapitation. Cerebral hemispheres were removed and homogenized in ice-cold 0.32 M sucrose, 5 mM HEPES–NaOH (pH 7.4) and 0.2 mM EDTA. Synaptosomes were prepared by differential and Ficoll-400 density gradient centrifugation of rat brain homogenate at 4 °C as described by CotmanSynaptic vesicles were isolated from nerve terminals of brain cerebral hemispheres using the hypo-osmotic shock  at 4 °C for 60 min. Synaptic membranes were isolated by centrifugation at 20,000 g for 30 min. After centrifugation of cytosolic fraction at 55,000 g for 60 min the supernatant was used to obtain the pellet of synaptic vesicles Micro-dissected cortices from one-day-old mouse pups were incubated with trypsin and DNAse I for 15 minutes at 37 °C. Before mechanical dissociation, cells were washed thoroughly and incubated for 15 min in inactivating solution . Obtained mass was triturated and passed through 40-μm nylon mesh cell strainer. Single cell suspension was washed and resuspended in plating medium. Cell count and viability were determined using haemocytometer and trypan blue exclusion assay.6 cell/mL) on poly-D-lysine-precoated 6-well plates. The preconditioned astrocyte feeder cultures for trophic support were seeded on porous polyester membrane of Transwell inserts with 0.4 μm pore size. Experiments were carried out after 14 days of co-culturing.Glial feeder cultures were prepared in advance from cortices of the newborn mouse pups using the trypsinization, adherence-based selection of astrocytes and generation of monolayers with approx 60% confluence. For co-culturing, primary neurons were plated . Platelets were used in the experiments for up to 3 hours after blood sampling without loss of response to activators .To exclude the activated platelet preparations based on dot plots of side scatter versus forward scatter and to evaluate the cell count the flow cytometry analysis was performed using COULTER EPICS XL flow cytometer . For each sample 20,000 events were acquired.+ for K+ was performed resulting in final K+ concentration of 70 mM. Synaptic vesicles were allowed to take up the dye (5 μM) for 20 min before being washed twice. Before the initiation of destaining the steady-state level of fluorescence was registered to exclude the spontaneous exocytosis. Destaining was calculated in percentage of total accumulated dye.Synaptic vesicle recycling in primary cultured neurons was registered with fluorescent styryl dye FM1-43 that reversibly binds to the neuronal plasma membrane and becomes trapped within endosomes/recycled synaptic vesicles during endocytosis. To stimulate vesicle turnover and dye uptake the isosmotic substitution of NaCells were loaded with ratiometric indicator FURA-2/AM (1 μM) or Fluo-3/AM (4 μM) at 30 °C for 60 min. BAPTA/AM loading started 30 min after FURA-2/AM loading. Then, cells were washed and left for 30 min to let complete de-esterification. Measurements were performed using the QuantaMaster 40 spectrofluorimeter .t/F0). Background values were obtained after permeabilization of the cells with digitonin and washout of Fluo-3 at the end of experiments. In situ calibrations were conducted in the presence of 5 μM A23187. The intracellular [Ca2+] was calculated according to the Grynkiewicz, Poenie and Tsien equationIn dual-excitation mode FURA-2-loaded samples were excited alternately at 340 and 380 nm and fluorescence was recorded at 510 nm to calculate the 340/380 excitation ratio. Fluo-3-loaded samples were excited at 506 nm and the fluorescence intensity changes were recorded at 526 nm. The intensity of Fluo-3 was normalized to baseline after background subtraction (F2+] in intracellular stores primary cultured neurons and platelets were loaded with Rhod-2/AM (4 μM) or Mag-Fluo-4/AM (5 μM) for 1 h at 30 °C and then left to de-esterify in dye-free medium for 2 h. Rhod-2-loaded samples were excited at 530 nm and the fluorescence intensity was recorded at 580 nm. Mag-Fluo-4-loaded samples were excited at 480 nm and the fluorescence intensity was recorded at 520 nm. The intensities were normalized to baseline after background subtraction.For measurements of [Ca2+]i in Fluo-3-loaded platelets the specimens were excited at 488 nm and emission was collected through a 505-nm long pass filter.For confocal imaging platelets were loaded with Fluo-3/AM, Rhod-2/AM and Mag-Fluo-4/AM as described in previous section. The staining with anti-CD63-APC was carried out at 4 °C for 30 min. Platelets were then washed, plated on slides and viewed under the confocal laser scanning microscope LSM 510 META, Carl Zeiss, with objective Plan-Apochromat 100 × /1.4 Oil DIC at 37 °C. For registration of the changes in [CaThe specimens with anti-CD63-APC-labeled platelets were excited with 633-nm laser and emission was collected through a 650-nm long pass filter. Cortical neurons loaded with Rhod-2/AM were excited with 543-nm laser and emission was collected using a 560–615 nm band-pass filter. Mag-Fluo-4-loaded cells were excited at 488 nm and emission was collected using a 505–530 nm band-pass filter.Images and time series were captured with high-resolution digital camera AxioCam HRc. Acquisition software: Zeiss LSM Image Browser Version 4.0.0.241.The changes in the acidification of intracellular compartments were registered with pH-sensitive fluorescent dye acridine orange (AO) that tends to form dimers and multimers after protonation at acidic pH. This results in decrease of green fluorescence that is inherent for monomeric dye. Platelets/synaptosomes/isolated synaptic vesicles or neurons were preincubated at 30 °C for 10 min. Fluorescence intensity measurements were started with application of AO (5 μM) and recorded at excitation and emission wavelengths of 490 and 530 nm, respectively. The 4-AP was applied at the steady-state level of dye accumulation. The curves were normalized to the similar traces in the absence of cells/synaptic vesicles according to Equation 1:Ft and F0 are the fluorescence intensities of acridine orange with and without cells/synaptic vesicles, respectively.where Ft and F0 are the fluorescence intensities of rhodamine 6G with and without cells, respectively.The changes of plasma membrane potential were measured using the potentiometric fluorescent dye rhodamine 6G. Cortical neurons were preincubated at 30 °C for 10 min and the measurements were started by application of rhodamine 6G at a final concentration of 0.5 mM. Fluorescence intensity was recorded at excitation and emission wavelengths of 528 and 551 nm, respectively. The 4-AP was applied at the steady-state level of dye accumulation. To estimate the changes of membrane potential the curves were normalized to the similar traces in the absence of cells according to Equation 1, where The release of endogenous glutamate was registered using the glutamate dehydrogenase assay36+ (1 mM) in cuvette under continuous stirring condition. Endogenous glutamate release was registered with excitation at 340 nm and emission at 460 nm. At the end of measurements different concentrations of exogenous glutamate were applied for calibration of the fluorescent signal.Platelets/synaptosomes/neurons/synaptic vesicles  were incubated at 30 °C for 10 min with glutamate dehydrogenase (20 U/mL) and NADn independent experiments. The significant difference between the means was detected by one-way ANOVA at the 0.05 significance level. The absence of prominent exocytotic peak (25–30% from accumulated dye) typical for functional synaptosomes and neurones (insets of Data are presented as means with standard error of the mean (s.e.m.) for How to cite this article: Kasatkina, L. A. 4-Aminopyridine sequesters intracellular Ca2+ which triggers exocytosis in excitable and non-excitable cells. Sci. Rep.6, 34749; doi: 10.1038/srep34749 (2016).


Drosophila phototransduction is mediated by phospholipase C, leading to activation of transient receptor potential (TRP) and TRP-like (TRPL) channels by mechanisms that are unresolved. A role for InsP3 receptors (IP3Rs) had been excluded because IP3R mutants (itpr) appeared to have normal light responses; however, this was recently challenged by Kohn et al. , who reported defects in phototransduction after IP3R-RNAi knockdown. They concluded that InsP3-induced Ca2+ release plays a critical role in facilitating channel activation, and that previous failure to detect 3RIP phenotypes resulted from trace Ca2+ in electrodes substituting for InsP3-induced Ca2+ release. In an attempt to confirm this, we performed electroretinograms, whole-cell recordings, and GCaMP6f Ca2+ imaging from both IP3R-RNAi flies and itpr-null mutants. Like Kohn et al., we used GMRGal4 to drive expression of 3R-RNAiUAS-IP, but we also used controls expressing GMRGal4 alone. We describe several GMRGal4 phenotypes suggestive of compromised development, including reductions in sensitivity, dark noise, potassium currents, and cell size and capacitance, as well as extreme variations in sensitivity between cells. However, we found no effect of IP3R RNAi or mutation on photoreceptor responses or Ca2+ signals, indicating that the IP3R plays little or no role in Drosophila phototransduction. Drosophila is mediated by phospholipase C (PLC), culminating in activation of TRP channels, but how PLC is coupled to channel activation is unresolved. A recent study reported phototransduction defects after InsP3 receptor RNA interference (IP3R-RNAi), supporting a critical role for InsP3-induced Ca2+ release. However, we found that phototransduction was quantitatively unaffected not only after IP3R-RNAi, but also in IP3R-null mutants. Instead, we describe novel phenotypes in photoreceptors from flies expressing the transcription factor  used to drive RNAi expression, which potentially account for the reported defects. The results indicate that IP3R plays no significant role in Drosophila phototransduction while emphasizing the need for caution when using Gal4 drivers.Phototransduction in microvillar photoreceptors such as those of Drosophila, there are two such “light-sensitive” channels, encoded by the transient receptor potential (trp) and trp-like (trpl) genes. Both are permeable to Ca2+, with TRP being particularly Ca2+ selective (PCa:PNa ∼50:1) and contributing the majority of the light-induced current  from the microvillar membrane (Microvillar photoreceptors respond to light using G protein–coupled phospholipase C (PLC) cascades, leading to activation of nonselective cation channels . In Dros current . First c current  and iden current  more thaeceptors , a role or IP3R; . Subsequor IP3R;  and its or IP3R; , or PIP2 protons . Our ownmembrane .3Rs played no role in Drosophila phototransduction was seriously challenged by a recent study using RNA interference  were reared in the dark at 25°C on standard medium . The wild-type strain was Oregon; for some experiments white-eyed mutants (1118w) were used, which are indistinguishable in terms of whole-cell electrophysiology, though more sensitive in ERG recordings.Flies ; referred to as GMR.1118; Spw/Cy; +,GMRGal4},UASP{w-wRNAi is referred to as GMRw (third chromosome); one copy of GMRGal4, UAS-wRNAi renders eye color almost white  despite presence of the wild-type w+ gene or multiple mini w+ transgenes (provided by A. Huber).1118w;;+ UASP{w-3R-RNAiIP} (third chromosome VDRC stock 6486); homozygous stock appears wild-type in ERG and whole-cell recordings.1118;P{w+,UAS-GCaMP6f}w .1118;P{w+,ninaE-GCaMP6f}w transgenic flies expressing GCaMP6f in photoreceptors R1-6 under control of the Rh1 opsin (ninaE) promoter: second and third chromosome lines made in-house using the pCaSpeR4 vector and GCaMP6f cDNA from Addgene : expresses near normal PLC protein levels (80%) but has <10% catalytic activity due to a point mutation (Ser347Ala) in the catalytic site, and another (Thr1007Ser) in the C terminus (90B.0l(3)itpr larval lethal, null mutation of IP3R due to small deletion; referred to as itpr itpr  were crossed to+, ey-Gal4,UAS-FLP}/CyO;P{ry+,FRT82B}P{w+, GMR-hid},3CLR/TM6yw;P{w . F1 non-Cy and non-TM6 then have itpr-null mosaic eyes (aic eyes .To generate whole-eye IP+w) FRT82B, itpr/TM6 flies were crossed to yw,EGUF;FRT82B flies and recordings made from red-eyed  itpr mosaics, using sibling itpr/TM6, red-eyed wild-type, and wild-type mosaic eyes  as controls. Further combinations using crosses from these parent lines are described in the text.To obtain similar eye pigmentation for ERGs, red-eyed (m): 120 NaCl, 5 KCl, 10 N-Tris-(hydroxymethyl)-methyl-2-amino-ethanesulphonic acid (TES), 4 MgCl2, 1.5 CaCl2, 25 proline, and 5 alanine, pH 7.15. For Ca2+ free solutions, CaCl2 was omitted and 1 mm Na2EGTA was added. Other solutions are described in text. The intracellular pipette solution was (in mm): 140 K gluconate, 10 TES, 4 Mg-ATP, 2 MgCl2, 1 NAD, and 0.4 Na-GTP, with or without 1 or 2 mm K2EGTA, pH 7.15. Chemicals were obtained from Sigma-Aldrich. Recordings were made at room temperature (21 ± 1°C) at –70 mV  using electrodes of resistance ∼10-15 MΩ. Series compensation of >80% was applied when required for macroscopic currents, but not for sampling quantum bumps and dark noise. Data were collected and analyzed using Axopatch 200 or HEKA amplifiers and pCLAMP v. 9 or 10 software (Molecular Devices). Quantum bumps and spontaneous dark events were analyzed using Minianalysis (Jaejin Software), analyzing typically at least 50–100 bumps/events per cell. Photoreceptors were stimulated via a green (540-nm) ultrabright light-emitting-diode (LED) controlled by a custom-made LED driver; intensities were calibrated in terms of effectively absorbed photons by counting quantum bumps at low intensities.Whole-cell patch clamp recordings of photoreceptors from dissociated ommatidia from newly eclosed, dark-reared adult flies of either sex were performed as previously described e.g.  on inverw+} transgenes  meant it was not always possible to obtain strictly identical pigmentation. However, the use of red light minimizes any effect of variable eye color . The maximum intensity (100 on figures) corresponded to ∼107 effectively absorbed photons per photoreceptor per second in white-eyed wild-type flies (1118w). Signals were amplified by a DAM60 DC preamplifier (WPI) and sampled and analyzed using pClamp software (Molecular Devices).ERGs were recorded as described previously e.g.,  from fliin vivo from intact flies via the deep pseudopupil (DPP). Excitation light (470 nm) was delivered from a blue power LED (Cairn Research), and fluorescence of whole ommatidia (via 40× oil objective) or DPP (20× air objective) was measured via a photomultiplier tube (Cairn Research) using 515 nm dichroic and OG515 longpass filters. Background fluorescence was subtracted using estimates from identical measurements from flies lacking fluorescent constructs. For dissociated ommatidia, ΔF/F0 was calculated using the F0 value measured in Ca2+-free solution (see above). To minimize any adverse long-term effects of exposure to Ca2+-free solution  and hence to maximize the chance of detecting any Ca2+ release, ommatidia, plated in normal bath, were individually perfused by a nearby (∼10–20 µm) puffer pipette and measurements made within ∼20–40 s of perfusion onset, always confirming that a normal, rapid (latency <10 ms), and large fluorescence signal was recovered on return to normal (1.5 mm Ca2+) bath. The 1 or 2 s blue excitation light used to measure the fluorescence is a supersaturating stimulus and sufficiently bright to photoisomerize 100% of the visual pigment molecules at least once, reaching a photoequilibrium with ∼70% metarhodopsin. After the first measurement  the ommatidium was briefly exposed to intense, photoequilibrating red  illumination to reconvert metarhodopsin to rhodopsin, returned to the control solution (1.5 mm Ca2+), and allowed to dark-adapt for at least 2 min before the next measurement. The effective intensity of the green illumination for in vivo measurements from the DPP was calibrated in effectively absorbed photons by measuring the rate at which it converted metarhodopsin to rhodopsin, as previously described  and 5′-GAACTCCACCTTCGGAATCA-3′ (reverse). The housekeeping gene Drosophila Ef1a48D primers, TCCTCCGAGCCACCATACAG (forward) and GTCTTGCCGTCAGCGTTACC (reverse), were used as internal controls.Total RNA was extracted by RNeasy Micro kit (Qiagen). Ten to twenty retinae were collected as described above for each biological group and homogenized by the TissueLyser (Qiagen) with six Zirconia 1 mm beads (Thistle) for 50 s twice, the samples were passed to the Qiashredder column (Qiagen), and standard procedures from RNeasy micro kit were followed. All nucleic acid preparations were quantitated by absorbance measurements at 260 nm using a NanoDrop instrument. The quantitative real time qRT-PCR was performed with a SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen) and ABI 7500 fast instrument (Applied Biosystems). The levels of InsPm Tris, 1 mm EDTA, 25 mm NaCl, and 200 µg/ml fresh Proteinase K and homogenized by the TissueLyser with nine Zirconia 1-mm beads for 50 s four times. The samples were then incubated at 37°C for 30 min and 95°C for 3 min (to destroy the proteinase K). The qPCR was set up with Terra qPCR Direct SYBR Premix (Takara) alongside either the IP3R primers, 5′-AAAATGCGTAGCATCGCTCT-3′ (forward) and 5′-CACCACCGGCTTTAGTTGAT-3′ (reverse), or the primers from the Rp132 housekeeping gene, 5′-CAAGAAGCTAGCCCAACCTG-3′ (forward) and 5′-CACTCACCGACAGCTTAGCA-3′ (reverse).Quantitative PCR experiments on genomic DNA (gDNA) were also conducted using the ABI 7500 fast instrument. Ten retinae were collected for each biological group, and gDNA was extracted with 10 m3R-RNAiUAS-IP transgene, single flies were homogenized by the gDNA extraction buffer described above, and the following primers (sequences from VDRC) were used to perform the gDNA PCR: 5′-CGCGAATTCCCTTCGCCAGAGCGTGGAAA-3′ (forward), 5′-CGCTCTAGAACGCCAACATTGCGGAGCAG-3′ (reverse), and 5′-CACAGAAGTAAGGTTCCTTCACAAAGATCC-3′ (reverse).For verifying the presence of the t tests or one-way ANOVAs with posttests as specified in the text and figure legends) were performed in GraphPad Prism5.Statistical tests (two-tailed 3R expression was suppressed by 3R-RNAiUAS-IP (two copies in most experiments), driven by one copy of GMRGal4. However, in most cases, controls from flies expressing GMRGal4 alone (without 3R-RNAiUAS-IP) were lacking. This is an important control, because GMRGal4 homozygotes expressing two copies of GMRGal4 have severe degeneration and developmental phenotypes , as well as the second chromosome GMRGal4 line  but without UAS-wRNAi. We used the same UAS-3RIP-RNAi line as 3IPR-RNAi transgene in all the backgrounds used by gDNA PCR of diagnostic sequences (from VDRC website). Effective knockdown of IP3R mRNA was validated by qRT-PCR of dissected retinal tissue, with 32.8% ± 1.7%  IP3R mRNA remaining compared with GMR/+ controls. Approximately half of this (∼15%) is likely attributable to contaminating tissue in the dissected retinae , it was apparent that GMRGal4/+ photoreceptors did not have the usual wild-type adult appearance. Ommatidia with one copy of GMRGal4, irrespective of 3RIP-RNAi, were shorter than in wild-type (∼65 vs. ∼85 µm) and often had a less well-formed appearance , again irrespective of 3RIP-RNAi (A) encoded by the Shaker gene, which typically reaches ∼4 nA (at 20 mV), vs. 2–3 nA for the delayed rectifier encoded by Shab , which appeared to show an approximately twofold reduction in QE in recordings made with EGTA (n = 8). This was marginally significant (p = 0.02) on a direct t test, but not on an ANOVA including 3R-RNAiGMRw/IP data with and without EGTA (p = 0.18). In all lines, inclusion of EGTA in the pipette increased bump amplitude, presumably due to suppression of the negative feedback effects of Ca2+ on the bump wave form , i.e., the fraction of incident photons evoking a quantum bump, which is determined by the amount of visual pigment (rhodopsin) and the probability that a photoisomerized rhodopsin successfully generates a quantum bump. One of the key findings in lectrode A–D. One ave form E. This wGMRGal4 was a large variability in QE . Although the majority of cells had QE values clustering within ∼20–60% of wild-type values, cells were regularly encountered (11/78 cells) in which QE was at least 10-fold, and sometimes >100-fold, lower (3RIP-RNAi appeared not to make a difference (5/11 cells were from GMR or GMRw controls without 3RIP-RNAi). In certain experiments , 3RIP-RNAi flies when recorded with EGTA. Given that we were unable to replicate these results even using IP3R-null mutants (see below), one possible explanation for their findings is the fortuitous inclusion of data from such insensitive cells.A conspicuous feature of recordings from flies carrying one copy of d, lower D. Such ld, lower . AlthougGMRGal4/3RIP-RNAi flies had reduced levels of spontaneous activity even without EGTA in the electrode. However, we found that these spontaneous dark events were greatly reduced in frequency in GMRGal4/+ photoreceptors irrespective of 3RIP-RNAi , whole-cell recordings from wild-type photoreceptors exhibit an ongoing spontaneous barrage of miniature (∼2-pA) bump-like events occurring at rates of ∼2/s, and which are believed to arise from spontaneous activation of G proteins . Kohn etP3R-RNAi F, G, sugGMRGal4 displayed a number of phenotypes suggestive of compromised development. These are presumably caused by adverse, nonspecific effects of Gal4 in the developing eye and seem potentially able to account for many, if not all, of the results reported by 3RIP-RNAi knockdown using either one or two copies of 3R-RNAiUAS-IP. Unfortunately, the GMRGal4,UAS-wRNAi fly stock used by 3RIP-RNAi flies would still have shown some phenotypes when compared with appropriate GMRGal4 controls. Therefore, for further analysis, we turned to IP3R-null mutants itpr, referred to as itpr) are larval-lethal; however, whole-eye null mosaics can be generated by inducing mitotic recombination in the developing eye using the flippase-flippase recognition target (FLP-FRT) system under control of ey-Gal4 . Previously, we found that itpr-null eye mosaics expressed no detectable IP3R protein and only trace amounts of genomic DNA or mRNA, from contaminating material in the dissections used to isolate retinal tissue , attributable to contaminating  tissue in dissected retinae .Null mutations of the IP ey-Gal4 . As contl tissue . Because retinae . We alsoitpr eye mosaics were indistinguishable from controls in intracellular recordings , but not compared with recordings from wild-type mosaic eyes .Previously, it was reported that photoreceptor responses from cordings  or wholecordings . Howeverp = 0.8, . By implitpr mosaic eyes revealed clear abnormalities, usually being noticeably larger and more bulbous than wild-type eyes and typically containing a variable number of irregular or darkened facets  and were of normal length, and in whole-cell recordings, the photoreceptors had capacitances and potassium channel profiles (not shown) similar to wild-type , QE appeared to be unaffected in either control flies or itpr-null mosaics  is approximately two orders of magnitude higher than for Sr2+ (∼30 µm), this should reduce trace Ca2+ to low nm levels while leaving ∼1.5 mm free Sr2+  reduction in QE. Because of the difficulty of unequivocally identifying bumps (and hence accurately estimating QE) under these conditions, we also simply recorded macroscopic responses to brief flashes. Peak amplitudes of responses were reduced approximately three- to fivefold after 2- to 3-min perfusion with the EGTA buffered Sr2+ solution, and time to peak slowed from ∼80 ms to >200 ms. Again, however, there was no difference between itpr-null mosaics and controls . Although we do not exclude the possibility of two sites, we do not believe that this can be concluded from the effects of Sr2+. When trace Ca2+ is buffered with EGTA, our results indicate that Sr2+ influx is also much less effective than Ca2+ influx in supporting light-induced bump amplification (whether in controls or itpr-null mutants), and hence the most parsimonious explanation would be that the same site could also be responsible for the reduction in dark events.Previously, 2+ release from IP3Rs is critical in facilitating phototransduction, then the simplest and most direct test for revealing its role should be testing sensitivity to light in Ca2+-free bath, an experiment not performed by 2+-free bath were unaffected in itpr-null mosaics  via a puffer pipette, peak responses were slowed and amplitude was reduced approximately fivefold, which is slightly more severe than the approximately threefold reduction previously reported with normal electrode solutions  was reduced by ∼100-fold (itpr/+ siblings) or itpr-null mosaics.If Ca mosaics ; howeverns e.g., . With ti100-fold . However3R-null mutants (itpr), we were unable to detect any of the phenotypes described by 3R knockdown in 3RIP-RNAi flies. Furthermore, in contrast to the variability we encountered in ERG recordings from itpr mutants or whole-cell recordings from GMRGal4 flies, we found no such variability in responses from single photoreceptors (with or without EGTA), despite making recordings from >60 cells from >30 flies (relative SD for QE ∼0.2–0.25 in both mosaics and controls). This reinforces our view that the variable ERG phenotypes in itpr-null mosaics  have essentially no light response  with H43norpA flies carrying one copy of GMRw with and without UAS-3RIP-RNAi.Although null mutants of PLC  appeared rather sensitive to genetic background. In the F1 of three different crosses to introduce one copy of GMRw into a H43norpA background . The response amplitudes in H43norpA;bw/+;GMRw/H43st, norpA;bw/Sp;GMRw/st, or H43norpA;bw/Cy;GMRw/st, which represent the genotypes closest to H43norpA;3R-RNAi/stGMRw/bw; IP flies of 3RIP-RNAi knockdown, but according to our results it appears attributable to one copy of GMRGal4.ERGs from ckground , ERG amp3RIP-RNAi in whole-cell patch-clamp recordings from H43norpA. Once again, in recordings made with electrode solution without EGTA, they reported no significant difference between H43norpA and H43norpA;GMRw/3RIP-RNAi flies, with both having a similar ∼4-log unit reduction in sensitivity compared with wild type. By contrast, when using EGTA-buffered electrode solution, H43norpA cells were reported to be unaffected, whereas sensitivity in H43norpA;3RIP-RNAi was further drastically reduced, such that cells were essentially completely unresponsive to the brightest lights. However, when we recorded from H43norpA photoreceptors  we found that 1 mm EGTA in the electrode already eliminated or drastically reduced sensitivity to light  gave no response at all to 100× brighter flashes (∼106 photons).to light D, E. ThuGMRGal4 significantly suppressed the ERG in H43norpA but found no additional effect of 3RIP-RNAi, whereas in whole-cell recordings, we already found a profound suppression of sensitivity by EGTA in H43norpA. It is difficult to explain the failure of H43norpA control flies. However, we found that H43norpA photoreceptors can be very sensitive to facilitation, and the few cells (3/31) that failed to show suppressed sensitivity using EGTA in the electrode had a substantial leak currents or low-resistance gigaseals, both likely permeable to Ca2+.In summary, we found that one copy of 2+ indicators in dissociated ommatidia show that the Ca2+ signal in response to blue excitation light (a supersaturating stimulus) is dominated by massive Ca2+ influx via light-sensitive channels , this residual “Ca2+-free” signal was reported to be unaffected in itpr-null mosaic mutants, suggesting it was not due to InsP3-induced Ca2+ release from internal stores  for a comprehensive picture  or itpr-null mutations  of EGTA.Although our measurements in the presence of Ca2+ rise in vivo in completely intact flies by monitoring GCaMP6f fluorescence in the deep pseudopupil  were somewhat less sensitive than wild type  of the InsP3 receptor on the light response of Drosophila photoreceptors. An apparent exception was the compromised ERG in at least some itpr-null mosaic eyes , we found no effect of 3R-RNAiIP or itpr-null mutation on photoreceptor sensitivity in vivo  reductions in QE irrespective of 3RIP-RNAi  to influence the onset of the electrical light response, which has a latency of <10 ms and peaks within ∼100 ms even under Ca2+-free conditions at these intensities (e.g., 2+ indicator dyes (itpr-null mutation or 3RIP-RNAi knockdown (3-induced release from internal stores. Previously, we found that this signal all but disappeared in the absence of extracellular Na+ and suggested that the rise might be due to reequilibration of Na+/Ca2+ exchange in response to the massive light-induced Na+ influx that persists under these conditions (2+, but not influx, was required for the Ca2+ rise in Ca2+-free solutions. However, in a recent study (2+-free rise dependent on Na+ influx, but it was also eliminated in mutants of the Na+/Ca2+ exchanger and accelerated by overexpression of the exchanger, strongly supporting our original suggestion.Although a clear Caes e.g., . As prevtor dyes , we alsonockdown  and is tnditions . This want study , we foun3RIP-RNAi or itpr-null mutants either in vivo or in whole-cell recordings with or without EGTA in the electrode, and together with a recent study (3-induced release of Ca2+ from internal stores. Our results therefore support earlier conclusions that the IP3R plays no significant role in the light response in Drosophila photoreceptors (GMRGal4/+ flies suggestive of compromised development, which we attribute to pleiotropic effects of Gal4 expression in the developing eye, and which should be carefully controlled for in any experiments making use of this widely used driver.In conclusion, we were unable to find any phototransduction phenotypes in nt study , we founeceptors . We have


The n-terminal tail of histone H4 recruits repair proteins, including 53BP1, to DNA double-strand breaks (DSB) and undergoes dynamic acetylation during DSB repair. However, how H4 acetylation (H4Ac) recruits repair proteins and reorganizes chromatin during DNA repair is unclear. Here, we show that the bromodomain protein BRD2 is recruited to DSBs. This recruitment requires binding of BRD2’s tandem bromodomains to H4Ac, which is generated at DSBs by the Tip60/KAT5 acetyltransferase. Binding of BRD2 to H4Ac protects the underlying acetylated chromatin from attack by histone deacetylases and allows acetylation to spread along the flanking chromatin. However, BRD2 recruitment is spatially restricted to a chromatin domain extending only 2 kb either side of the DSB, and BRD2 does not spread into the chromatin domains flanking the break. Instead, BRD2 facilitates recruitment of a second bromodomain protein, ZMYND8, which spreads along the flanking chromatin, but is excluded from the DSB region. This creates a spatially restricted H4Ac/BRD2 domain which reorganizes chromatin at DSBs, limits binding of the L3MBTL1 repressor and promotes 53BP1 binding, while limiting end-resection of DSBs. BRD2 therefore creates a restricted chromatin environment surrounding DSBs which facilitates DSB repair and which is framed by extensive ZMYND8 domains on the flanking chromatin. This fundamental reorganization of chromatin is required to promote access of the repair machinery to the DSB and to facilitate repair processes such as end-resection and homology search during homologous recombination. The repair of DSBs therefore requires a tightly coupled partnership between chromatin remodeling complexes and the DNA repair machinery.The repair of DNA double-strand breaks (DSBs) requires recruitment of DNA repair proteins to the site of damage and is linked to changes in nucleosome dynamics and histone modification2. These repressive factors include HDACs and the NuRD remodeling complex3, HP1 and the H3K9 methyltransferase SUV39H16 and histone demethylases7. H2A.Z is also exchanged onto the chromatin at DSBs (by NuA4-Tip609), creating H2A.Z-nucleosomes with an enlarged acidic patch. The unacetylated H4 tail then binds to the acidic patch9, promoting nucleosome interaction and formation of compact chromatin at the DSB11. This repressive chromatin may restrict local nucleosome motion, limit transcription and erase local epigenetic modifications which may inhibit repair12. These temporary repressive structures are dismantled by removal of H2A.Z9 and exchange of other histone variants at DSBs15. This promotes H2A ubiquitination17 and acetylation of H4 (by KAT5/Tip6019). Together, these histone modifications create an open, acetylated chromatin structure which is required for efficient DSB repair20.DSB repair promotes a rapid, PARP-dependent recruitment of repressive complexes to the chromatin, followed by a coordinated shift to a more open, flexible chromatin conformation21, is recruited to DSBs through dual binding to H2A ubiquitinated at lysines 13 and 15 (H2AK13/15)23 and H4K20me224. H2A is ubiquitinated by RNF168 in response to DNA damage23, whereas 53BP1 relies mainly on pre-existing H4K20me2. However, 2 proteins which bind to H4K20me2, the repressor L3MBTL125 and the lysine demethylase KDM4A26, can block 53BP1 loading at DSBs. Consequently, the VCP/p97 ATPase complex is recruited to DSBs where it actively removes L3MBTL1 and promotes 53BP1 binding25. Further, acetylation of H4 at lysine 16 (H4K16Ac) can limit interaction of 53BP1 with H4K20me2 and regulate 53BP1 recruitment to DSBs27. The H4 tail, and access to H4K20me2, is therefore critical for facilitating H4 acetylation, the loading of 53BP1 and for the correct processing of the chromatin during DSB repair.The H4 tail functions as a central hub for regulating recruitment of DSB repair proteins to the site of damage. 53BP1, which plays a central role in directing repair to either the HR or NHEJ repair pathways27. Studies have shown that the acetylated H4 tail provides a binding surface for a number of bromodomain proteins29. This includes ZMYND8, which recruits the repressive NuRD complex to DSBs and promotes transcriptional silencing during repair30 and BRD4, which may limit spreading of DNA damage signals along the chromatin31. However, how histone acetylation signatures created by DNA damage recruit specific bromodomain proteins to DSBs is currently unknown. Here, we show that the bromodomain protein BRD2 is rapidly recruited to H4Ac at DSBs where it forms a spatially restricted domain extending 2 kb either side of the break. BRD2 therefore defines a novel, spatially restricted chromatin domain which surrounds the DSB and is essential for DSB repair.The H4 tail contains multiple lysine residues which are acetylated by NuA4-Tip60 in response to DNA damage32. To determine if BRD2 was recruited to DSBs, Zinc Finger Nucleases (ZFNs) were used to create DSBs in the non-essential PPP1R12C gene (p84-ZFN)8 or at 2 intergenic sites at position 46.125 Mb (K230-ZFN) and 31.22 Mb (M15-ZFN) on chromosome 333. BRD2 was monitored by Chromatin Immunoprecipitation (ChIP) and real-time quantitative PCR using a validated BRD2 antibody  blocked BRD2 recruitment to DSBs  increased H4Ac, and this increase was also lost when BRD2 was silenced with siRNA  should lead to less mobile chromatin. H4Ac weakens histone-DNA interactions, leading to an increase in histone solubility when nuclei are extracted in high salt after DSB production10. Bleomycin, which creates DSBs, increased the NaCl solubility of histone H3 . The HDAC inhibitor TSA did not alter DSB production by p84-ZFN , with low level accumulation of L3MBTL1 at several sites on the flanking chromatin Fig. . UnexpecNext, we examined how BRD2 contributes to L3MBTL1 eviction at DSBs. Because loss of BRD2 leads to rapid deacetylation of H4 Fig. , we exam45 from occupying the unacetylated H4 tail during DSB repair. This favors the recruitment of BRD2, which binds to H4K8Ac/K12Ac36, creating H4Ac/BRD2 domains which resist HDAC attack and further limit L3MBTL1 binding to H4K20me2. In the absence of H4Ac/BRD2 (or VCP/p97) L3MBTL1 binds to H4K20me245 on the unacetylated H4 tail  or VCP/p97 activity (with NMS873) blocked 53BP1 loading at DSBs Fig. . Importame2 Fig. . In the 38, which may contribute to the higher radiosensitization by JQ1 compared to NMS873 or shRNA to BRD2. Cells treated with JQ1 or NMS872 also retained large numbers of γH2AX foci post-irradiation  to insulate H4Ac domains from HDACs, including HDAC complexes recruited to DSBs50, indicates the importance of maintaining H4Ac during repair. Intriguingly, BRD2 is required for spreading of H4Ac away from the DSB and loading of ZMYND8, indicating that BRD2 is upstream of ZMYND8. How localization of BRD2 to the DSB promotes spreading of H4Ac and ZMYND8 far beyond the restricted BRD2 domain is unclear. Previous work has shown that several DNA damage induced modifications, including γH2AX51 and H3K9me34, are initiated at the DSB and then spread linearly away from the site of damage. BRD2 may bind to nascent H4Ac at the DSB, protecting it from HDAC activity and facilitate spreading of H4Ac along the damaged chromatin. In this case, BRD2 may promote spreading of H4Ac by simply binding to and shielding H4Ac from HDACs. In addition, BRD2 may also alter the local nucleosome organization at the DSB to promote access to the H4 tail by the Tip60 acetyltransferase. In the absence of BRD2, local H4Ac is reduced (and exposed to HDAC activity), and H4Ac fails to spread, preventing both BRD2 and ZMYND8 loading. However, given the complexity of ZMYND8’s histone reader activity, which includes both histone acetylation and methylation binding domains42, the loss of ZMYND8 binding and H4Ac in the absence of BRD2 may not simply reflect the loss of H4Ac spreading from the break. As an alternative, BRD2 may promote editing of other histone modifications at the DSB which may influence ZMYND8 binding. For example, removal of H3K4me3 may be required for binding of ZMYND8 to the damaged chromatin42. If H3K4 demethylation also requires either BRD2 or H4Ac, then loss of BRD2 would prevent H3K4me3 demethylation and loading of ZMYND8. This would leave any nascent H4Ac on the flanking chromatin exposed to HDACs and therefore lead to loss of both ZMYND8 and H4Ac on the flanking domains. A more detailed analysis of histone methylation and acetylation signatures surrounding DSBs is needed to address this issue.A key function for recruitment of BRD2 to nascent H4Ac was to protect the underlying H4Ac from attack by HDACs. Hyperacetylated chromatin domains are required for DSB repair52. Our results now demonstrate that both BRD2/H4Ac and VCP/p9725 work together to prevent L3MBTL1 from accumulating on H4K20me2 at DSB. This co-operation is largely mediated by H4Ac, since loss of BRD2 is rescued by HDAC inhibition. We propose that acetylation of histone H4 limits L3MBTL1 binding to H4K20me2 and thereby favoring its removal by VCP/p97. This is likely to be important during the initial nucleosome processing which occurs immediately after DSB production. During DSB repair, there is a transient nucleosome compaction mediated by binding of the unacetylated H4 tail to the acidic patch on adjacent nucleosomes12, followed by nucleosome reorganization and release of the unacetylated, H4K20me2 tail from the nucleosome surface  and VCP/p97’s ATPase activity are therefore essential to prevent L3MBTL1 from occupying H4K20me2 on the unacetylated H4 tail. This creates stable H4Ac/BRD2 domains which are resistant to HDAC attack and which block L3MBTL1 binding to the adjacent H4K20me2. In the absence of either H4Ac/BRD2 or VCP/p97  crystal violet and colonies with >50 cells scored visually as previously described54. Protocols for transfection, establishing cell lines, western blot analysis (including antibodies) and sequence of siRNA and shRNA constructs is described in the supplementary methods section.U2OS, HEK293T and HeLa cells  were maintained in Dulbecco’s Modified Eagles Medium supplemented with 10% Fetal Bovine Sera55, HEK293 cells expressing the alt-NHEJ reporter48 or HeLa cells expressing an NHEJ reporter48 were treated with DMSO, JQ1 or NMS-873. 24hr later, cells were transfected with I-Sce I or eGFP (control) plasmid using Lipofectamine 2000 . GFP positive cells were detected 48hr later using the BD LSR II cell analyzer  and data analyzed with the BD Diva software package. Data were normalized to eGFP (control) transfected cells to correct for transfection efficiency.U2OS cells expressing the DR-GFP reporterHEK293T cells were transfected with p84-ZFN or other ZFNs and allowed to recover for 18 hr. Cells were fixed in 1% methanol-free formaldehyde for 10 min to crosslink proteins, lysed in ChIP buffer , sonicated, and cleared by centrifugation. Part of the supernatant was digested with proteinase K (65 °C for 2 hr), the DNA isolated by spin columns and input DNA quantitated by Real Time PCR. Equivalent amounts of chromatin were incubated with primary antibody (overnight at 4 °C) followed by protein G agarose beads precoated with sperm DNA. Immune complexes were washed in low and high salt ChIP buffers , eluted, incubated in NaCl (65 °C for 2 hrs) and digested with proteinase K. Purified DNA was quantitated by RT-qPCR using the Step One Plus real time PCR system . PCR protocols, primer pairs and ChIP grade antibodies are listed in supplementary methods.10. Genomic DNA prepared for ChIP was amplified using primer pairs located either side of the DSB (supplementary methods) by real time qPCR and the percent of DSBs estimated by the change in signal resulting from cleavage of the DNA. 18s rRNA genomic DNA signal was used to ensure equal input DNA.DSB production was monitored using standard PCR based techniques previously described by us56. Standard controls included immunoprecipitation with IgG, which yielded essentially no signal. Relative fold enrichment was optimized to the input control and expressed as IP/Input DNA. The relative increase in signal after cutting by p84-ZFN, K230 or M15 was calculated as [IPZFN/InputZFN]/[IPControl/InputControl]. All ChIP assays were repeated at least twice , with individual RT-qPCR reactions carried out in duplicate  and the results presented ± standard deviation.PCR amplification  utilized 95 °C for 5 mins, followed by 33 cycles of: 30 sec @ 95 °C/ 30 sec @ 60 °C/ 30 sec @ 72 °C and a final extension step of 5 mins @ 72 °C. Serial dilutions of the starting material were used to determine the linear range of PCR amplification prior to use. 18S rRNA genomic sequences were used to standardize input genomic DNACells were fixed with PBS/paraformaldehyde (4%) 0–30 minutes after irradiation. Time between initial exposure and termination by fixation was <2 minutes. Cells were permeabilized in methanol, washed in PBS and incubated in Triton X-100 (0.2%) for 5 minutes. Cells were then washed twice in PBS and blocked with fetal bovine serum (10%) for 20 minutes. Slides were incubated with primary and secondary antibody with washing between each step, mounted with Fluoromount-G  and imaged with a Zeiss AxioImager Z1 microscope equipped with an Axiocam MRc Rev.3 Color Digital Camera and Plan APO 63X/1.4 oil M27 lens . Acquisition software and image processing utilized the Zeiss AxioVision software package . Cells with >5 foci were scored as positive.2, 0.1% Triton) containing 1.0 M NaCl for 40 minutes at 4oC with agitation as previously described10. Cells were collected by centrifugation at 100,000 g (Beckmann Ultracentrifuge) for 20 minutes, and the supernatant retained for western blot analysis.Washed cells were resuspended in buffer A (20 mM Hepes pH7.9, 0.5 mM DTT, 1 mM PMSF, 1.5 mM MgClAll data generated or analyzed during this study are included in this published article and the associated supplementary data files.Supplementary information


Phosphoinositide-3-kinase (PI3K) is an enzyme group, known to regulate key survival pathways in acute myeloid leukaemia (AML). It generates phosphatidylinositol-3,4,5-triphosphate, which provides a membrane docking site for protein kinaseB activation. PI3K catalytic p110 subunits are divided into 4 isoforms; α,β,δ and γ. The PI3Kδ isoform is always expressed in AML cells, whereas the frequency of PI3Kγ expression is highly variable. The functions of these individual catalytic enzymes have not been fully resolved in AML, therefore using the PI3K p110δ and p110γ-targeted inhibitor IPI-145 (duvelisib) and specific p110δ and p110γ shRNA, we analysed the role of these two p110 subunits in human AML blast survival. The results show that PI3Kδ and PI3Kγ inhibition with IPI-145 has anti-proliferative activity in primary AML cells by inhibiting the activity of AKT and MAPK. Pre-treatment of AML cells with IPI-145 inhibits both adhesion and migration of AML blasts to bone marrow stromal cells. Using shRNA targeted to the individual isoforms we demonstrated that p110δ-knockdown had a more significant anti-proliferative effect on AML cells, whereas targeting p110γ-knockdown significantly inhibited AML migration. The results demonstrate that targeting both PI3Kδ and PI3Kγ to inhibit AML-BMSC interactions provides a biologic rationale for the pre-clinical evaluation of IPI-145 in AML. Although clinically, morphologically and biologically acute myeloid leukaemia (AML) appears to represent a heterogeneous group of diseases, the disease seems to rely on common intra-cellular survival and self-renewal pathways downstream of the driver oncogene . It is ePI3K is an enzyme group that generates phosphatidylinositol 3, 4, 5-triphosphate which provides a membrane docking site for the serine-threonine kinase Protein Kinase B (PKB), also known as AKT . Many ofClass I PI3K catalytic p110 subunits are divided into 4 isoforms; α, β, δ and γ. Within the p110 catalytic subunit the p110δ isoform is always expressed in AML cells , 15. ConTo determine the level of detectable PI3Kδ and PI3Kγ in AML we examined five AML cell lines and eight primary AML samples. Figure The next step was to determine the effect of PI3Kδ and PI3Kγ inhibition on AKT activation. AML cell lines and primary tissue were treated with increasing concentrations of IPI-145 and cultured for 4 hours before Western blot analysis was performed. PI3Kδ and PI3Kγ inhibition showed a reduction in AKT phosphorylation at serine 473 (s473) in all AML cell lines and primary AML tested. There was also a dose dependent reduction in MAPK phosphorylation in response to IPI-145 in OCI-AML3, MV4-11 cell lines Figure  and primThe microenvironment is an essential component underpinning survival of AML blasts. Inhibiting AML blast adhesion to BMSCs via VLA4-VCAM/fibronectin interactions via the PI3K/AKT signalling pathway is associated with improvements in chemotherapy-induced tumour cytotoxicity . TherefoIn order to determine whether PI3Kδ and PI3Kγ inhibition can disrupt AML migration we undertook assays using BMSC conditioned media (CM) as a migration stimulus and normal media (NM) as a control. Figure We next evaluated the genetic inhibition of PI3Kδ and PI3Kγ using shRNA in OCI-AML3. To do this we generated lentivirus-mediated PI3Kδ and PI3Kγ knockdowns. These constructs induced PI3Kδ and PI3Kγ knockdown confirmed for up to 7 days by real-time PCR Figure . Using tDespite the recognition that AML represents a morphologically and genetically diverse collection of tumours it is also apparent that these tumours rely on common programs of self-renewal. The PI3K/AKT pathway is known to be constitutively active in 50-80% of patients with AML and is associated with overall decreased survival , 30. AccIt has been reported that AML proliferation and survival can be inhibited by the pan PI3K inhibitor LY 294002 . MoreoveIn non-malignant myeloid cells both PI3Kδ and PI3Kγ have distinct roles. For example, in mast cells PI3Kδ is required for activation by SCF and IL-3, which induces mast cell proliferation and adhesion , while PAKT phosphorylation on s473 can be detected in up to 80% of AML patients while the information on AKT phosphorylation on t308 is less well characterised. A study by Gallay et al (2009), showed that 24 out of 53 AML patients had high AKT phosphorylation on t308 and this (but not AKT phosphorylation on s473) was associated with high-risk cytogenetics and predicted poor overall survival in AML . Our resThe interactions between tumour and its bone marrow microenvironment have an important role in regulating the survival and proliferation of AML blasts. We have shown that inhibition of PI3Kδ and PI3Kγ can disrupt adhesion of AML blasts to BMSC and fibronectin, and that pre-treatment with IPI-145 can overcome the chemotherapy resistance which BMSC have been shown to provide to AML blasts. Overall we have shown an important role for PI3K and downstream signalling in the AML / BMSC pro-survival interaction. As such we provide data that support the clinical evaluation of combined PI3Kγ and δ inhibition (with IPI-145) in human AML.Anti-phosphorylated and pan, AKT, and MAPK antibodies, as well as PI3Kγ were purchased from Cell Signalling Technology . Anti-CD34-PE, anti-CD90-FITC, anti-CD73-PE, anti-CD105-APC antibodies as well as SDF-1 were purchased from Miltenyi Biotec . Anti-PI3Kδ antibody was purchased from R&D systems . LY294002, CAL101, IPI-145, MK2206 and AZD6224 were obtained from Selleck Chemicals . All other reagents were obtained from Sigma-Aldrich , unless indicated.The AML-derived cell lines were obtained from the European Collection of Cell Cultures and DMSZ where they are authenticated by DNA-fingerprinting. In the laboratory they are used at low passage number for a maximum of six months post-resuscitation, testing regularly for Mycoplasma infection. AML blasts were obtained from patients' bone marrow or blood following informed consent and under approval from the UK National Research Ethics Service (LRECref07/H0310/146) Table Human bone marrow stromal cells (BMSC) were isolated by bone marrow aspirates from AML patients. Mononuclear cells were collected by gradient centrifugation and plated in growth medium containing DMEM and 20% FBS and 1% l-glutamine. The non-adherent cells were removed after 2 days. When 60%-80% confluent, adherent cells were trypsinised and expanded for 3-5 weeks. BMSCs were checked for positive expression of CD105, CD73, and CD90 and the lack of expression of CD45 and CD34 by flow cytometry as previously described .Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses were performed as described previously. Briefly, whole cell lysates were extracted and sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation performed . ProteinCells were treated with different doses of IPI-145 then viable numbers measured with Cell TiterGlo . For the AML-BMSC co-cultures AML cell viability was measured using trypan blue exclusion.3 to 5×104 cells) were plated in methylcellulose medium (R&D systems) and colonies were visualised, measured and counted after 10 days. For normal CD34+ CFU-GM, CFU-M, CFU-M and BFU-E was measured.Control CD34+ HSC and primary AML cells . The lower compartment contained 30μL of conditioned media or serum free media supplemented with 100 ng/ml SDF1 and the cells were applied to the upper compartment and allowed to migrate for 3 h. The amount of viable migrated cells was determined by counting using trypan blue exclusion and expressed as a percentage of the input.BMSCs were grown in 96-well plates. Primary AML cells were incubated with 2.5 μM calcein-AM for 1 h at 37°C and 5% CO2. The fluorescence-labelled AML cells were added into stromal cell coated 96-well plates and incubated for the indicated time points. Non-adherent calcein-labelled cells were removed by gently washing and adherent cells were quantified in a fluorescence multi-well plate reader. For AML blast adhesion onto fibronectin (FN), 96 well plates were coated with 10 mg/ml FN for 1 h before the fluorescence-labelled AML cells were added. Again non-adherent calcein-labelled cells were removed by gently washing and adherent cells were quantitated in a fluorescence multi-well plate reader.Total RNA was extracted from 5 × 105 cells using the Nucleic acid Prep Station from Applied Biosystems , according to the manufacturer's instructions. Reverse transcription was performed using the RNA polymerase chain reaction (PCR) core kit (Applied Biosystems). Relative quantitative real-time PCR used SYBR green technology  on cDNA generated from the reverse transcription of purified RNA. After pre-amplification (95°C for 2 minutes), the PCRs were amplified for 45 cycles (95°C for 15 seconds and 60°C for 10 seconds and 72°C for 10 seconds) on a 384-well LightCycler 480 (Roche). Each mRNA expression was normalized against GAPDH mRNA expression using the standard curve method.4 cells/well/0.5ml. Cells were infected with lentiviral stocks at an MOI of 15 in the presence of 8 μg/ml Polybrene. Transduced cells were analysed by real-time-PCR (Roche).OCI-AML3 were plated onto 12 well plates at 5 × 10We used the Mann-Whitney U test to compare results in control to treated groups. Results with P < 0.05 were considered statistically significant (*). We also use the Two-way ANOVA with Sidak's post test. Results with P < 0.05 were considered statistically significant (*). Results represent the mean ± SD of 4 independent experiments. For Western blotting, data are representative images of 3 independent experiments. We generated statistics with Graphpad Prism 5 software .


Excessive matrix Ca2+ concentrations, especially when coincident with oxidative stress, precipitate opening of an inner mitochondrial membrane, high-conductance channel: the mitochondrial permeability transition pore (mPTP). mPTP opening has been implicated as a final cell death pathway in numerous diseases and therefore understanding conditions dictating mPTP opening is crucial for developing targeted therapies. Here, we have investigated the impact of mitochondrial metabolic state on the probability and consequences of mPTP opening. Isolated mitochondria were energised using NADH- or FADH2-linked substrates. The functional consequences of Ca2+-induced mPTP opening were assessed by Ca2+ retention capacity, using fluorescence-based analysis, and simultaneous measurements of mitochondrial Ca2+ handling, membrane potential, respiratory rate and production of reactive oxygen species (ROS). Succinate-induced, membrane potential-dependent reverse electron transfer sensitised mitochondria to mPTP opening. mPTP-induced depolarisation under succinate subsequently inhibited reverse electron transfer. Complex I-driven respiration was reduced after mPTP opening but sustained in the presence of complex II-linked substrates, consistent with inhibition of complex I-supported respiration by leakage of matrix NADH. Additionally, ROS generated at complex III did not sensitise mitochondria to mPTP opening. Thus, cellular metabolic fluxes and metabolic environment dictate mitochondrial functional response to Ca2+ overload.Mitochondrial Ca The resulting proton electrochemical gradient (Δp), comprising a membrane potential (ΔΨm) and pH gradient, is necessary for the production of adenosine triphosphate (ATP) and metabolite transport through the inner mitochondrial membrane (IMM)3.Mitochondria are capable of oxidising numerous substrates based on availability and metabolic demand. The delivery of energetic substrates to mitochondria provides reducing equivalents required for serial reduction of electron transport chain (ETC) redox centres. These redox reactions are coupled to expulsion of protons from the matrix into the intermembrane space (IMS)5. Thorough understanding of the (patho)physiological conditions mediating these homeostatic outcomes is important to help develop new therapeutic agents for a number of diseases including Parkinson’s Disease and stroke8.The functions of mitochondria extend beyond that of cellular ATP biosynthesis. Indeed, mitochondria participate in multiple regulatory signalling pathways stimulated in response to both physiological and pathophysiological stimuli. As key regulators of cell death pathways, mitochondria also play a critical role in determining cell fate2+ uptake plays an important role in cellular homeostasis, being driven by the maintenance of ΔΨm9. The mitochondrial permeability transition pore (mPTP) is a presumed proteinaceous entity in the IMM. Pore opening has generally been attributed to a structural change in a protein embedded within the membrane, which, under other conditions, seems to usually perform a physiological role11. The precise molecular composition and identity of the mPTP is highly controversial but candidates include the adenine nucleotide translocase (ANT), the voltage dependent anion channel (VDAC), spastic paraplegia 7 (SPG7), phosphate carrier (PiC) and components of the ATP synthase17. Recent observations have further complicated structural understanding of the mPTP complex in that He el al., challenging previous claims18 recently demonstrated intact permeability transition in the absence of membrane and C-ring subunits of the ATP synthase19.Mitochondrial Ca21. Failure of cellular Ca2+ homeostasis and consequent mitochondrial Ca2+ overload is the principal trigger for mitochondrial mPTP opening22. High-conductance mPTP opening is associated with osmotic swelling, loss of IMM potential, uncoupling of oxidative phosphorylation and metabolic collapse25.To date, the only unequivocally identified component of the pore complex is cyclophilin D (CypD), a matrix cyclophilin that regulates pore opening, conferring sensitivity of the pore to inhibition by cyclosporin A (CsA)29. To explore the impact of different metabolic conditions on the probability of pore opening, fluorescence-based analysis coupled to high-resolution respirometry was used to simultaneously measure mitochondrial Ca2+ uptake, ΔΨm, reactive oxygen species (ROS) production and respiration. The threshold of mPTP opening was assessed under distinct metabolic conditions by measuring the Ca2+ buffering capacity of isolated mitochondria. Characterisation of the relationship between substrate utilisation, oxygen consumption and sensitivity to pore opening will reveal events that precede and follow mPTP opening. Here, we report variation in mitochondrial sensitivity to Ca2+-induced mPTP opening based on the source of electron flux through the respiratory chain, as driven by different metabolic substrates, identifying mechanisms by which energetic state determines the mitochondrial response to Ca2+ overload.As mPTP opening is a determinant of cell death in an ever-growing list of diseases, it is important for clinical benefit to understand pathways culminating in pore opening to identify effective interventions preventing or limiting pore opening2+ retention capacity protocol adapted to simultaneously monitor multiple parameters was established using the FLIPRTETRA high content kinetic assay system. Using this assay, mPTP opening was identified as the release of mitochondrially sequestered Ca2+, the failure of mitochondrial Ca2+ uptake  and the collapse of the mitochondrial membrane potential (measured using TMRM).Mitochondria are capable of metabolising several different metabolic substrates. To understand how substrate availability determines the probability of mPTP opening, a Ca2+ (10 µM) were effectively removed from the extra-mitochondrial solution by mitochondrial Ca2+ uptake  caused mPTP opening and simultaneous collapse of ΔΨm under all metabolic conditions  glutamate/malate, (2) succinate or (3) succinate/rotenone. Under all metabolic conditions, initial aliquots of added Ca. m Fig. . Greaterons Fig. . Howeverone Fig.  and c whone Fig. . A decreate Fig. : deviati glutamatm and Ca2+ uptake were performed in isolated mitochondria in response to Ca2+ overload, using the Oroboros Oxygraph 2 K high resolution respirometry system coupled with a fluorescence detection unit.We next aimed to understand the consequences of the loss of mitochondrial inner membrane integrity on respiratory chain electron flow and to explore how metabolic substrate selectivity influences the bioenergetic consequences of mPTP opening. Simultaneous recordings of respiration, ΔΨ2+ concentrations resulted in complete mitochondrial Ca2+ uptake , ΔΨm loss occurred after a Ca2+-induced increase in respiration, remaining sustained at an elevated respiratory rate under these conditions  or CsA to, respectively, block mitochondrial Ca2+ uptake or delay mPTP opening. Mitochondria were incubated with glutamate/malate and subjected to a Ca2+ retention capacity assay. Consistent with published data33, we observed a small but significant decrease in State 3 respiration using both CsA and RuR  but also by preventing mPTP opening via CypD inhibition  or spontaneously dismutates at a high rate. Additionally, H2O2 can be directly produced by some mitochondrial sites (i.e. flavins38). Therefore, we measured H2O2 generated by isolated mitochondria to assess overall rates of mitochondrial ROS (mtROS) generation.Mitochondria are an important source of cellular ROS, also implicated in sensitising mPTP formation to Ca2+ concentration (Fluo-4FF) and H2O2 production  were measured simultaneously in a Ca2+ retention assay protocol. The rate of baseline H2O2 production by mitochondria energised using glutamate/malate was significantly higher than that generated by succinate alone  from complex II to complex I41. Overall, however, the contribution of RET to the total AmpR signal was low (~10%), and the resulting feed forward rates of mtROS flux in succinate/rotenone-driven respiration were significantly lower than in mitochondria energised by glutamate/malate  which was attributed to Ca2+-mediated, CsA-sensitive transient uncoupling (see above). In contrast, although a Ca2+-induced sustained increase in respiratory rate was supported by succinate  and reverse  electron flows. Indeed, in healthy mitochondria (before mPTP opening), the rate of H2O2 production following uncoupling with FCCP was: (i) dose-dependently reduced when mitochondria were respiring on succinate alone and; (ii) dose-dependently increased when using glutamate/malate as substrates  and complex III inhibition on the FLIPRTETRA high content kinetic assay system. Antimycin A stimulated a large CsA- and RuR-sensitive increase in H2O2 production across all substrates , suggesting that rates of mtROS production from complex III are augmented by mPTP opening  the probability of mPTP opening is increased when respiration is supported by succinate compared to glutamate and malate, a phenomenon reduced by the presence of rotenone; (ii) the efficiency of succinate to induce mPTP opening correlates with succinate-induced RET through complex I and associated mtROS production, (iii) mPTP-induced depolarisation inhibits RET from succinate energised mitochondria; (iv) in the presence of succinate, respiration is maintained following mPTP opening and loss of mitochondrial membrane potential; (v) when respiration is supported by glutamate/malate, mPTP opening causes a transient increase in respiration followed by a decrease that can be restored by addition of NADH or succinate, consistent with respiratory inhibition due to the loss of matrix NADH; and (vi) although ROS are generated from complex III in the presence of both glutamate/malate and succinate/rotenone, this does not sensitise mPTP opening.We have investigated the effects of mitochondrial substrate availability on the probability of Caet al. demonstrated that mPTP opening and subsequent cytochrome c release are more sensitive to Ca2+ when mitochondria are oxidising FADH2-linked rather than NADH-linked substrates42. We have added to these observations to understand metabolic consequence of Ca2+-induced mPTP opening, investigating bioenergetic changes, mitochondrial Ca2+ uptake, ΔΨm and mtROS generation. We have confirmed that respiration supported by succinate sensitises mitochondria to Ca2+-induced mPTP opening. Inhibition of electron flux from complex I to ubiquinone (UQ) by rotenone decreased both baseline mtROS production and the efficiency of succinate-energised mitochondria to undergo mPTP opening. This suggests that ROS generation through complex I may define the sensitivity of mitochondria to Ca2+-induced mPTP opening under these conditions. It is noteworthy that reperfusion following ischemia , has been associated with succinate accumulation and increased ROS generation by RET43. Prevention of Ca2+-induced RET using rotenone reduces oxidative stress in succinate-energised mitochondria, likely being responsible for the decreased mitochondrial sensitivity to Ca2+-induced mPTP opening. In agreement with Chouchani et al. reverse flow of electrons through complex I contribute to mPTP opening43. Additionally, we have also determined that ROS production through reverse flow is membrane potential-dependent and thus must precede mPTP opening.Previously, Huang 2+-induced mtROS production in succinate-energised mitochondria. Since high ΔΨm is necessary to drive RET and the reduction of NAD+, the observed Ca2+-induced decrease in mtROS production following mPTP opening and membrane depolarisation is likely due to the cessation of RET46. Consistent with these observations, addition of the ETC uncoupler, FCCP to intact/healthy mitochondria also dose-dependently decreased mtROS production in mitochondria energised by succinate alone. In succinate-energised mitochondria, the FCCP-mediated decrease in mtROS production was observed at concentrations consistent with membrane depolarisation and increased oxygen consumption47, responses analogous to mPTP opening. Additionally, inhibition of mPTP opening, using CsA and RuR, prevented the Ca2+-induced decrease in mtROS, suggesting that mPTP opening following Ca2+ uptake contributes to decreased oxidative stress in mitochondria metabolising succinate alone. mPTP-induced depolarisation therefore inhibits RET and mtROS generation from succinate (similar to FCCP), correlating with a transient increase in ROS in ‘succinate only’-energised condition and suggesting a transient peak in ROS can be efficient in pre-sensitising mPTP to Ca2+.We observed only a transient increase in Ca2+ -induced mPTP opening was observed. However, addition of rotenone under succinate-driven respiration reduced the probability of mPTP opening, suggesting that RET through complex I sensitises to mPTP opening, while forward electron flow through Complexes I and III does not affect the process. As expected, we observed an antimycin A-dependent increase in baseline mtROS production. Electron carriers upstream of the point of antimycin A-sensitive inhibition become reduced causing a large increase in baseline H2O2 production in mitochondria metabolising all substrates48. The antimycin A-dependent increase in H2O2 production is both greater after mPTP opening and sensitive to CsA and RuR, suggesting mPTP opening mediates the augmented mtROS production. Together, these observations suggest that complex III appears the major source of mtROS production following mPTP opening and, although glutamate/malate and succinate/rotenone produce ROS on complex III, it is not a sensitising factor for stimulating mPTP opening. ROS may have differential effects when generated at the inner or outer face of the mitochondrial inner membrane. Superoxide generation from complex I is directed towards the matrix and complex III to both the matrix and IMS, suggesting that complex I is the most significant source of superoxide generation towards the mitochondrial matrix50. As superoxide does not cross membranes and is extremely reactive, it will initially react with components that are exposed in the environment next to the site of generation52. Microdomains therefore are highly relevant for ROS species as they have a limited diffusion range due to their high, localised reactivity.Despite Complex I-linked substrates generating higher mtROS at baseline, no increased sensitivity to Ca2+ preceding mPTP opening. Ca2+-induced stimulation of respiration has been proposed to occur through different mechanisms. Increased NADH production by Ca2+ sensitive dehydrogenases (pyruvate and oxoglutarate) of the TCA cycle53, Ca2+-mediated stimulation of succinate dehydrogenase54, or dissipation of the H+ gradient by futile Ca2+ cycling could augment electron flow through the respiratory chain55. However, under our conditions the Ca2+-induced respiratory increase was fully blocked both by CsA and RuR. Thus, we hypothesise that low-conductance, reversible mPTP opening, may be responsible for the respiratory increase observed following sub-threshold Ca2+ uptake57. Given the absence of effect of low concentration Ca2+ on ΔΨm, it is likely that these mild uncoupling events can be compensated for through increased respiration, as observed previously using FCCP at low concentration47. The lack of effect of Ca2+ on NADH provision to complex I may reflect that principal sites of NADH production using glutamate/malate are the Ca2+-insensitive glutamate and malate dehydrogenases and, additionally, that succinate dehydrogenase under these conditions has been shown to be inactivated by oxaloacetate59.Under all metabolic conditions, rates of mitochondrial oxygen consumption increased following initial additions of Cam. In contrast to succinate-energised mitochondria, where respiration is maintained following mPTP opening, in glutamate/malate-energised mitochondria, respiratory rates decreased following Ca2+-induced high-conductance mPTP opening. Soluble NAD+/NADH is lost through the mPTP to the extra-mitochondrial space, limiting electron flux from UQ to complex III60. mPTP opening and respiratory decline were prevented by CsA and RuR as soluble reducing equivalent are preserved within the matrix. Intriguingly, addition of exogenous NADH or succinate following complete mPTP opening and inner membrane depolarisation is able to recover and increase respiration. These observations are consistent with those following Ca2+ overload and mPTP opening in succinate-driven respiration. The flavoprotein pool, via which electrons pass from succinate, is inner membrane bound, therefore facilitating maintenance of respiration as succinate saturates the pool and respiratory rate hits maximum. In contrast to respiration driven by glutamate/malate at complex I, where soluble respiratory intermediate NADH is lost from the matrix. Previously, Hawkins et al. observed that supplementation by succinate could prevent rotenone/oligomycin- and hypoxia-induced mitochondrial depolarisation. In agreement with the data presented here, succinate failed to maintain ΔΨm following mPTP opening61.High-conductance mPTP opening is consistent with mitochondrial inner membrane uncoupling and loss of ΔΨ2+-induced mPTP opening. We have characterised key modulatory factors, including mitochondrial energetic status and Ca2+ concentration in both regulating propensity for mPTP opening and determining consequence of pore opening. Together, these data further add to understanding the patho effects of pore opening. For decades, mPTP opening has been recognised as a critical factor in cellular death across numerous diseases. Therefore, understanding the precipitating factors, the pathways culminating in pore opening and the downstream metabolic consequences will aid the rational design and development of therapeutics, with potential utility in treating multiple difficult-to-treat disease states.In summary, we investigated the role of substrate selectivity and utilisation in determining propensity for CaAll chemicals and compounds were purchased from Sigma-Aldrich , unless otherwise specified. Fluorescent probes  were from Life Technologies .Female Sprague Dawley rats (250–300 g) were from Charles River  and allowed to acclimatise to conditions for four days. Animals were euthanised by cervical dislocation followed by immediate removal of livers. Animal care and procedures were performed in accordance with UK Animals (Scientific Procedures) Act, 1986. Procedures were carried under a UK Home Office licence and studies were approved by Eisai’s Institutional Animal Care and Use Committee (IACUC).62. Briefly, fresh rat livers were washed and finely minced in ice-cold wash buffer . Tissue was homogenised using 10 strokes of a glass/Teflon potter and drill, set to 1600 rpm, in 5x tissue volume of complete homogenisation buffer  , cOmplete Protease Inhibitor™ , adjusted to pH 7.5). Homogenates were centrifuged at 800 g for 10 minutes at 4 °C, supernatants transferred to a clean tube and then centrifuged further at 10,300 g at 4 °C for 10 minutes. Mitochondrial pellets were surface-washed using complete homogenisation buffer and the final centrifugation step repeated. The pellets were re-suspended in complete homogenisation buffer and protein concentration determined by bicinchoninic acid assay (BCA) . Mitochondrial suspensions (50 mg protein ml−1) were snap-frozen in liquid nitrogen and stored at −80 °C until use. All mitochondrial preparations were maintained at −80 °C for up to 7 months. Prior to activity assays, frozen mitochondria were thawed by briefly placing vials in a 37 °C water bath and then kept on ice until required.Mitochondria were isolated in accordance with published protocols2+ retention capacity was used to assess in vitro sensitivity to Ca2+ of isolated mitochondrial preparations. Mitochondria were washed in ice-cold mitochondrial assay buffer  to remove residual EDTA and re-suspended  = 1 mg protein ml−1) in complete MAB. To remove any contaminating Ca2+, MAB was pre-treated with Chelex 100 resin  and resin removed through filtration.Assessment of CaComplete MAB containing 2x Fluo-4FF penta-potassium salt  was supplemented with either: (1) 20 mM L-glutamic acid, monosodium salt, FAC = 10 mM; 4 mM L-malic acid sodium salt, FAC = 2 mM, (2) 20 mM L-glutamic acid monosodium salt, FAC = 10 mM; 4 mM L-malic acid sodium salt, FAC = 2 mM; 6 mM NADH, FAC = 3 mM, (3) 20 mM succinate disodium salt, FAC = 10 mM or (4) 20 mM succinate disodium salt, FAC = 10 mM; 2 μM rotenone, FAC = 1 μM). Final pH of the solutions was confirmed to be 7.4 and adjusted where necessary using NaOH.TETRA, Molecular Devices, Sunnyvale, CA) over 35 minutes at room temperature. CaCl2  in MAB (2.5 μl additions to 40 μl) was repeatedly added at 3 minute intervals.Mitochondrial suspensions  and supplemented Fluo-4FF containing MAB  were dispensed into a clear-bottom, black-walled 384 well plate containing compound using a Multidrop Combi Reagent Dispenser  and incubated for 10 mins at room temperature. Extra-mitochondrial fluorescence (ex. 470–495/em. 515–575) was measured at 6 second intervals (FLIPRm was measured using tetramethylrhodamine methylester (TMRM), a voltage-sensitive cationic lipophilic dye, partitioning and accumulating in the mitochondrial matrix based upon the Nernst equation. When TMRM is loaded at relatively high concentrations (>100 nM63), fluorescence within the mitochondria is auto-quenched. Any disruption to mitochondrial function (e.g. membrane uncoupling or electron transport inhibition) dissipates ΔΨm with TMRM redistribution yielding an increase in fluorescence upon relief of auto-quenching.Mitochondrial ΔΨg at 4 °C for 10 minutes and re-suspended in ice-cold complete MAB to a final concentration of 2 mg protein ml−1  = 1 mg protein ml−1). MAB was pre-treated with Chelex 100 resin  to remove any contaminating Ca2+, and resin removed through filtration. MAB containing TMRM  was supplemented as for CRC assay. Mitochondrial suspension  and supplemented TMRM containing MAB  were dispensed into a clear-bottom, black-walled 384 well plate containing the compounds using a Multidrop Combi Reagent Dispenser  and incubated with the compounds for 10 mins at room temperature to allow TMRM equilibration. Fluorescence (ex. 510–545/em. 565–625) was measured at 6 second intervals  over 35 minutes at room temperature. CaCl2  in MAB (2.5 μl additions to 40 μl) was repeatedly added at 3 minute intervals.To remove residual EDTA, mitochondria were centrifuged at 10,300 2O2 production was determined using Amplex Red (AmpR), a dye reacting with 1:1 stoichiometry with H2O2 in a peroxidase-catalysed reaction to produce the highly fluorescent resorufin. Mitochondria were washed in ice-cold MAB to remove residual EDTA and re-suspended  = 1 mg protein ml−1) in complete MAB. MAB was pre-treated with Chelex 100 resin  to remove any contaminating Ca2+, and resin removed through filtration.H−1) was supplemented as above for CRC assay. Mitochondrial suspension  and supplemented AmpR/HRP containing MAB  were dispensed into a clear-bottom, black-walled 384 well plate containing compound using a Multidrop Combi Reagent Dispenser  and incubated with compound for 10 mins at room temperature. Fluorescence (ex. 510–545/em. 565–625) was measured at 6 second intervals  over 35 minutes at room temperature. CaCl2  in MAB (2.5 μl additions to 40 μl) was repeatedly added at 3 minute intervals.MAB containing 2x AmpR (20 μM) and horseradish peroxidase  in MAB supplemented with either: (1) 10 mM L-glutamic acid, monosodium salt; 2 mM L-malic acid sodium salt, (2) 10 mM succinate disodium salt or (3) 10 mM succinate disodium salt; 1 μM rotenone. Mitochondrial suspensions were added to each chamber, maintained at 25 °C under constant stirring (250 rpm), Oxygraph 2 K calibrated using oxygen solubility factor 0.92 and respiration allowed to stabilise.Mitochondrial oxygen consumption was measured using the Oxygraph 2 K . Mitochondria were washed in ice-cold MAB, to remove residual EDTA. Mitochondria were re-suspended (1 mg protein ml−1) were added to chambers for parallel measurement of ΔΨm, extracellular Ca2+ and H2O2 respectively. A sequential titration protocol, including additions of 10 mM succinate disodium salt, 1 μM rotenone, 2.5 μM antimycin A, 0.5 μM carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP) and 3 mM NADH disodium salt, 0.5 μM CsA and 0.5 μM RuR were added, as indicated in results. Extra-mitochondrial Ca2+ fluorescence , ΔΨm and H2O2  were measured in parallel to respiration at 3 second intervals over the assay duration. CaCl2  in MAB (2 μl additions to 2 ml) was repeatedly added at 1.5 minute intervals.The Oxygraph 2 K was equipped with an O2K fluorimeter and fluorescence control unit  and TMRM (2 μM), Fluo-4FF (1 μM) and Amplex Red/HRP (10 μM/1 U mlP < 0.05.Data are presented as mean ± standard deviation (s.d.). Normalisation of data allowed for control of inter-assay variability. AUC was calculated using ScreenWorks . Curve fitting used GraphPad Prism version 7.02 for Windows . Statistical significance tests performed are indicated in figure legends. Statistical significance was determined as Supplementary Information