In pulmonary artery smooth muscle cells (PASMCs) it has been proposed that nicotinic acid adenine dinucleotide phosphate (NAADP) triggers increases in cytoplasmic Ca2+ via L-SR junctions, in a manner that requires initial Ca2+ release from lysosomes and subsequent Ca2+-induced Ca2+ release (CICR) via ryanodine receptor (RyR) subtype 3 on the SR membrane proximal to lysosomes. L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy, which has restricted detailed investigations of the junctional coupling process. The present study utilizes standard and tomographic transmission electron microscopy to provide a thorough ultrastructural characterization of the L-SR junctions in PASMCs. We show that L-SR nanojunctions are prominent features within these cells and estimate that the junctional membrane separation and extension are about 15 nm and 300 nm, respectively. Furthermore, we develop a quantitative model of the L-SR junction using these measurements, prior kinetic and specific Ca2+ signal information as input data. Simulations of NAADP-dependent junctional Ca2+ transients demonstrate that the magnitude of these signals can breach the threshold for CICR via RyR3. By correlation analysis of live cell Ca2+ signals and simulated Ca2+ transients within L-SR junctions, we estimate that “trigger zones” comprising 60–100 junctions are required to confer a signal of similar magnitude. This is compatible with the 110 lysosomes/cell estimated from our ultrastructural observations. Most importantly, our model shows that increasing the L-SR junctional width above 50 nm lowers the magnitude of junctional [Ca2+] such that there is a failure to breach the threshold for CICR via RyR3. L-SR junctions are therefore a pre-requisite for efficient Ca2+signal coupling and may contribute to cellular function in health and disease.Herein we demonstrate how nanojunctions between lysosomes and sarcoplasmic reticulum (L-SR junctions) serve to couple lysosomal activation to regenerative, ryanodine receptor-mediated cellular Ca Nevertheless there is now a growing recognition of the widespread occurrence and functional significance of cytoplasmic nanospaces in cells from species across several kingdoms9.The importance of cytoplasmic nanojunctions to cellular signaling and thus to the modulation of cell function was recognised several decades ago11.In this respect, membrane-membrane junctions between lysosomes and the sarcoplasmic reticulum (L-SR junctions) are of particular interest; not least because they have been hypothesized to couple calcium signaling between these two organelles2+ signaling was uncovered through early studies on the Ca2+ mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP)12, which demonstrated that NAADP released Ca2+ from a store other than the sarco/endoplasmic reticulum (S/ER)13, that could then trigger further Ca2+ release from the S/ER by Ca2+- induced Ca2+ release (CICR)17. A major advance in our understanding was then provided by the demonstration that the NAADP-released Ca2+ was from an acidic lysosome-related store19 in a manner that requires two pore segment channel subtype 2 (TPC2)20. However, studies on pulmonary arterial smooth muscle cells (PASMCs) had also identified a significant specialization, namely L-SR nanojunctions. It was hypothesized not only that these nanojunctions were necessary for coupling between lysosomes and the SR, but that they could both coordinate and restrict their relationship to the SR by preferentially targeting ryanodine receptors while excluding inositol 1,4,5-trisphosphate (IP3) receptors11. Importantly, NAADP-dependent Ca2+ bursts primarily arise from lysosomes in the perinuclear region of PASMCs and appear to promote rapid, local Ca2+ transients that are of sufficient size to activate clusters of SR resident ryanodine receptor subtype 3 (RyR3) and thus initiate, in an all-or-none manner, a propagating global Ca2+ wave11.That L-SR junctions may play an important role in cellular Ca21. However, L-SR junctions may also make as yet unforeseen contributions to vascular pathologies as highlighted by the fact that Niemann-Pick disease type C1 results, in part, from dysregulation of lysosomal Ca2+ handling22 and is known to precipitate portal hypertension23, while other lysosomal storage diseases  accelerate pulmonary arterial hypertension25. Moreover, observed hypertension is often associated with dysfunction of cholesterol trafficking26, increased plasma cholesterol levels, vascular lesion formation, atherosclerosis/thrombosis and medial degradation27. Therefore lysosomal Ca2+ signaling is of considerable clinical interest. That L-SR junctions may be of further significance to pathology is also evident, for example, from the fact that in the pulmonary artery smooth muscle L-SR junctions underpin Ca2+ waves initiated by endothelin 1, the levels of which are elevated in pulmonary hypertension, systemic hypertension and atherosclerosis29. An understanding of how specific Ca2+ signals are functionally initiated therefore has important translational implications.The specialization of the proposed L-SR junction is likely important in smooth muscle cell physiology, e.g., in vasomotion, and in the recycling of organelles and programmed cell death by autophagy2+ regulation has been of current interest in several recent research and review articles , yet a quantitative elucidation of the mechanism by which Ca2+ signals are generated by the endolysosomal system is still lacking. Given the likely importance of L-SR junctions to Ca2+ signaling in health and disease, we sought to determine whether this nano-environment would indeed be able to effectively generate the previously observed NAADP-induced Ca2+ signals. In particular, we set out to verify the following hypotheses in PASMCs: (1) L-SR nanojunctions should be observable in the ultrastructure of these cells, (2) NAADP induces sufficient Ca2+ release from the lysosome to initiate activation of RyR3 embedded in the junctional SR, and (3) the combined effect of activation of L-SR nanojunctions in a cytoplasmic “trigger zone” determines the threshold of global [Ca2+]i for the biphasic release process.Lysosomal Ca2+ signals, accurate investigation of dynamic signaling within these spaces cannot be achieved with currently available instrumentation. To overcome this issue, we took an integrative approach by combining our own electron microscopy of L-SR nanojunctions, existing kinetic data on the Ca2+ transporters and buffers, and prior knowledge of the NAADP-induced Ca2+ signal features into a quantitative model of a typical L-SR nanojunction. This model is based on stochastic simulations of intracellular Ca2+ diffusion by Brownian motion implemented using the particle simulator MCell37.Due to the minute spatial scale of the nanojunctions generating the primary Ca2+ to activate CICR via RyR3 clusters embedded in the junctional SR. Perhaps most importantly, we show that L-SR coupling is determined both by the integrity of L-SR junction on the nanoscale and the quantal summation of Ca2+ release from multiple, activated junctional complexes.In the present manuscript we have verified the existence of L-SR nanojunctions within the ultrastructure of PASMCs, and shown that lysosomes can release sufficient CaAll the experiments and procedures were carried out in accordance with the guidelines of the University of British Columbia Animal Care Committee, and in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. The Research Ethics Committees from both these institutions specifically and independently approved this study.2, 10 mM HEPES, 0.05 mM CaCl2, and 10 mM glucose . After extraction of the heart and lungs and their placement in HEPES buffer, several rings from the primary and secondary branches of the pulmonary artery were dissected and immediately immersed in fixative solution. The primary fixative solution contained 2.5% glutaraldehyde  in 0.1 M sodium cacodylate buffer  at room temperature. The artery rings were then washed three times in 0.1 M sodium cacodylate . In the process of secondary fixation, the tissue rings were fixed with 1% OsO4  in 0.1 M sodium cacodylate buffer for 1 h followed by three 10-minute washes with distilled water anden bloc staining with 2% uranyl acetate . In the final process of dehydration, the samples underwent 3 washes in 100% ethanol. The artery rings were then resin-infiltrated in increasing concentrations  of a 1:1 mix of Epon and Spurr’s resins . The infiltration process was completed by three passages in 100% resin. All of the ethanol dehydration and resin infiltration steps were carried out by using a laboratory microwave oven . The blocks were finally resin-embedded in molds and polymerized overnight in an oven at 60°C.An adult male Wistar rat  was anesthetized with 3 ml of Thiotal . For the purpose of this phase of the study, one representative healthy animal usually suffices. This choice also minimizes unnecessary animal sacrifice. The thoracic cavity was opened and flooded with warm physiological saline solution (PSS) containing 145 mM NaCl, 4 mM KCl, 1 mM MgClFor standard (2D) electron microscopy imaging, 80-nm sections were cut from the embedded sample blocks on a Reichert Ultracut-E microtome  using a diamond knife  and were collected on uncoated 100- and 200- mesh copper grids . The sections were post-stained with 1% uranyl acetate  and Reynolds lead citrate  for 12 and 6 minutes, respectively. Electron micrographs at various magnifications were obtained with a Hitachi 7600 transmission electron microscope  at 80 kV.Lysosomes in these images were identified according to their well established appearance and features: they are bound by a single lipid-bilayer membrane, with a granular, more or less uniform luminal matrix that is more electron dense than the surrounding cytosol. Secondary lysosomes may also contain less granular structures within the finer matrix. Moreover, lysosomes are normally distinguishable from endosomes by their larger size, hence we set a threshold “diameter” of > 200 nm for acceptance of a lysosome, below which all vesicles were excluded.To obtain electron microscopic tomograms, we cut 200-nm-thick sections from the same sample blocks used for standard imaging. The sections were then collected on Formvar  coated slot copper grids  and post-stained with 1% uranyl acetate and Reynolds lead citrate for 20 and 10 minutes, respectively. We surveyed the sample sections using a FEI Tecnai G2 200 kV transmission electron microscope  and identified regions of interest containing L-SR junctions. We then acquired tomograms of several of those regions by taking 2D scans through the sample every 5° of inclination as it was tilted between −60° and +60° with respect to horizontal. The scans were reconstructed with the Inspect3D  software tools and structures of interest, primarily lysosomal and SR membranes in the same cellular neighbourhood, were traced out using Amira  software.The images of the samples were further processed using GIMP  to enhance membrane contrast in the interest of improving our characterization of the L-SR junctions.http://www.inkscape.org), converting the pixel measurements to nm using the scale bar in the recorded micrographs. By modifying the Inkscape script for measuring lengths, we were able to output the measurements directly into a text file, which we used to produce the histograms inhttp://www.gnuplot.info) to produce the histograms and plots presented herein.The SR and lysosomal membranes were outlined, highlighted and measured in pixels using the Inkscape software program  of the following composition (mM): 130 NaCl, 5.2 KCl, 1 MgCl2, 1.7 CaCl2, 10 glucose, and 10 HEPES, pH 7.45 . Single arterial smooth muscle cells were isolated from second-order branches of the pulmonary artery. Briefly, arteries were dissected out and placed in low Ca2+ solution of the following composition (mM): 124 NaCl, 5 KCl, 1 MgCl2, 0.5 NaH2PO4, 0.5 KH2PO, 15 NaHCO3, 0.16 CaCl2, 0.5 EDTA, 10 glucose, 10 taurine and 10 HEPES, pH 7.4. After 10 min the arteries were placed in the same solution containing 0.5 mg/ml papain (Fluka) and 1 mg/ml bovine serum albumin (Sigma) and kept at 4°C overnight. The following day 0.2 mM 1,4-dithio-DL-threitol (Sigma) was added to the solution, to activate the protease, and the preparation was incubated for 1 h at room temperature (22°C). The tissue was then washed at 3°C in fresh low Ca2+ solution without enzymes, and single smooth muscle cells were isolated by gentle trituration with a fire-polished Pasteur pipette. Cells were stored in suspension at 4°C until required.To obtain sufficient cell isolation to appropriately test significance, 1–3 adult male Wistar rats  were sacrificed by cervical dislocation. The heart and lungs were removedµM Fura-2-AM in Ca2+-free PSS in an experimental chamber on a Leica DMIRBE inverted microscope and then superfused with Fura-2 free PSS for at least 30 min prior to experimentation. Intracellular Ca2+ concentration was reported by Fura-2 fluorescence ratio . Emitted fluorescence was recorded at 22°C with a sampling frequency of 0.5 Hz, using a Hamamatsu 4880 CCD camera via a Zeiss Fluar 40×, 1.3 n.a. oil immersion lens and Leica DMIRBE microscope. Background subtraction was performed on-line. Analysis was done via Openlab imaging software .PASMCs were incubated for 30 min with 5I = 0) as described previously17. The pipette solution contained (in mM): 140 KCl, 10 HEPES, 1 MgCl2 and 5µM Fura-2, pH 7.4. The seal resistance, as measured using an Axopatch 200B amplifier , was ≥ 3 GΩ throughout each experiment. Series resistance and pipette resistance were ≤ 10 MΩ and ≤ 3 MΩ, respectively. All experiments were carried out at room temperature (≈ 22°C).NAADP was applied intracellularly in the whole-cell configuration of the patch-clamp technique, and in current clamp mode (The main stages of the quantitative modeling approach are:1.The design of 3D software mesh objects (nets of interconnected triangles by which surfaces can be represented in computer graphics) representing a typical L-SR region, including a whole lysosome and a portion of neighbouring SR network. These objects are built to-scale following the ultrastructural characterization of the L-SR junctional regions as it results from our electron microscopy image analysis; this phase was carried out using the “3D content creation suite” Blender ;2.2+ and mobile Ca2+ buffers);The positioning of the relevant transporters on the reconstructed membranes  according to information gathered from the literature on their typical membrane densities and the implementation of the transporter's known kinetics and multi-state models and of the ion diffusivities (Ca3.http://www.mcell.org)37. In a nutshell, MCell reproduces the randomness of the molecular trajectories, of the ion transporter flickering and of the relevant chemical reactions by probabilistic algorithms governed by pseudo-random number generators . This enables the simulation of a number of microphysiological processes, all stochastically different from one another. The average outcome of the processes, to mimic the instrumental output during experimental measurements, is obtained by taking the average of a desired quantity, e.g., [Ca2+], over a large number of simulations all initiated with a different seed;The simulation of molecular Brownian motion in the cytosol by random walk algorithms; this phase was performed by writing appropriate code for the stochastic particle simulator MCell -SR junctions that are pivotal in the process of SR Ca2+ refilling during asynchronous [Ca2+] waves39. We furthermore hypothesize that in order for these nanojunctions to appropriately regulate Ca2+ signaling, they must be separated by a distance of approximately 20 nm and be of the order of a few hundred nm in lateral dimensions, as inferred from previous studies on PM-SR junctions11.The example record in40. In standard (2D) transmission electron microscopy (TEM) images, lysosomes typically appear as elliptical structures bound by a single lipid bilayer, a feature that distinguishes them from mitochondria. Depending on the lysosomal system stage, they also tend to have a more or less uniformly electron-dense interior as compared to the surrounding cytosol41. They can be distinguished from endosomes by their larger size and darker lumen and they differ from peroxisomes, since the latter usually display a geometrically distinct and markedly darker structure called “crystalloid” in their luminal area. Moreover, it would appear that peroxisomes are found far more frequently in liver, kidney, bronchioles and odontoblasts than in other cell types42 .To identify lysosomes, SR regions and L-SR nanojunctions, we recorded and surveyed 74 electron micrographs of rat pulmonary arterial smooth muscle taken from samples prepared as described in the Materials and methods section. The images in43, we have excluded organelles  with such characteristics from our statistical count.Occasionally, organellar remnants are still visible inside these ovals, a characteristic that identifies them as multi-vesicular bodies “MVB” in. As it iFrom each of the relevant smooth muscle regions surveyed, we obtained high-resolution images of areas containing lysosomes and L-SR junctions in order to quantitatively characterize them  in17, corresponding to [Ca2+]i at time point 1 in the example record shown in17). This leads to a ∆[Ca2+]shoulder of approximately 300 nM17. Let us now estimate the number of lysosomes required to generate a ∆[Ca2+]shoulder of such magnitude in a PASMC, given the following assumptions:To understand the generation of the signal “shoulder”, such as that observed at time point 1 in the lower panel of1.2+] of a lysosome, [Ca2+]lys, is in the range of 400–600µM, as determined in mouse macrophages44 and that it is homogeneous across the lysosomal population;That the luminal [Ca2.2+ release rate during bursts is ≈ 106 ions/s  and that it becomes negligible for values of [Ca2+]lys below ≈ 80µM, as suggested by the single channel kinetics of TPC2 signaling complexes in lipid bilayer studies45;That the Ca3.V = 2.2 × 10−17 L;That lysosomes are spheroids with dimensions gathered from our EM characterization see, and hen4.Vcyt = 2.4 × 10−12 L by modeling a cell as a 130-µm-long cylinder, 6µm in diameter46, and accounting for nuclear, SR, mitochondrial and lysosomal volumes as described in the supplementary material.That a smooth muscle cell cytosolic volume can be calculated as2+]lys = (400 to 600)µM − 80µM = 3.2 × 10-4 M to 5.2 × 10−4 M into the cytosol . Each lysosome contribution to [Ca2+]i, ∆[Ca2+]i,lys, can then simply be calculated by taking the lysosome-to-cell volume ratio into account:With these assumptions accepted, from points 1. and 2. above we gather that a lysosome can release a potential ∆[Ca2+]shoulder asTherefore, we can calculate the number of lysosomes that may contribute to the magnitude of ∆[Ca2+]shoulder of 300 nM, which is typically observed during the localized Ca2+ release phase of the NAADP-induced Ca2+ signal.In summary, between 60 and 100 lysosomes would be necessary (and possibly sufficient) to provide a ∆lys determined in macrophages, and it is plausible that the total releasable [Ca2+]lys in a PASMC may differ from that value and that it may also vary over the lysosome population. Lastly, the uncertainty in the number of lysosomes/cell emerging from the electron micrographs as described above may also contribute to this discrepancy.How many lysosomes do we actually expect to be in a PASM cell of our sample tissue? We can obtain a rough estimate of this number from the TEM imaging we performed for this study. In each 80-nm-thick TEM sample section, we see 5–10 lysosomes/cell. Lysosomes are predominantly localized to the perinuclear region of the cytoplasm, but seldom in the subplasmalemmal area, consistent with previous observations by optical microscopy11, Ca2+ bursts activate SR resident RyR3 channels within L-SR junctions and thus initiate a propagating Ca2+ wave by CICR. The stochastic simulations developed here attempt to reproduce the phenomenon of the generation of [Ca2+] transients within individual L-SR junctions, considering the Ca2+ release kinetic requirements for lysosome-resident TPC2 signaling complexes and the rate of Ca2+ capture by the SERCA2a localized on the neighbouring SR membrane47, and to link these junctional transients to the observed bursts.In the hypothesized model outlined previously2+ signal within the nanospace of a representative L-SR junction as faithfully as possible.The thorough quantitative image analysis described in the previous section yields critical information for our first modeling phase, in which we built a dimensionally accurate virtual lysosome and a portion of the SR system, closely apposing the lysosome , so as t2+ current in isolated lysosomes48, by the single channel conductance45, thus estimating that a typical lysosomal membrane may contain ≈ 20 TPC2. To obtain this value, we used experimental condition data48 to extract values for the ionic potential,Eion, across the lysosomal membrane. We then employed the authors’ current-voltage (I-Vm) data to compute whole-lysosome conductance values (gWL) as a function of the applied membrane potential,Vm, from Ohm’s law:I =gWL(Vm −Eion). We report details on this calculation in the supplementary material.From the available literature we estimated the number of TPC2 and SERCA2a likely distributed on the lysosome and SR membranes, respectively. We obtained the former number by dividing the macroscopic whole-lysosome conductance, calculated from the current values reported in a recent study on TPC2-mediated Ca/μm239. Other input data for the model are the estimates of lysosomal volume and of the [Ca2+]lys, from which we calculated the actual number of ions in the lysosome prior to the beginning of Ca2+ release (see previous section). This, in turn, was used in the extrapolation of the TPC2 complex Ca2+ release rate as a function of time, as follows.Moreover, we have estimated the density of SERCA2a on the SR within L-SR junctions to be equivalent to that previously predicted for PM-SR junctions as approximately 62502+ conductance of TPC2 signaling complex provides valuable information regarding its biophysical properties and, importantly, highlights the fact that, for a given relatively low activating concentration of NAADP 45. We used the Ca2+ conductivity measured in that study and values of the lysosome membrane potential49 to calculate the maximal Ca2+ current,Imax, as 2.4 × 106 ions/s. We then applied a weighted quadratic fit to the channel’s open probability (Po) as a function of the reported [Ca2+]lys data with constraints that thePo would tend to zero at [Ca2+]lys = 80µM, based on the observation that below [Ca2+]lys = 100µM essentially no single channel openings were observed45. Another constraint for the fit was that the curve be within the standard deviation value ofPo at the highest reported [Ca2+]lys = 1 mM. From the quadratic fit, we then obtained our ownPo-vs-[Ca2+]lys table Po ×Imax until the luminal concentration reaches the next lower point in the table, and so on untilPo = 0 at [Ca2+]lys = 80µM. The Ca2+ release rate as a function of time obtained in this manner is shown inA recent electrophysiological study of the Ca50, which was further informed by more recent isoform-specific studies52. In particular, since we are interested in simulating the SERCA2a Ca2+ uptake only in terms of its influence on the shaping of the junctional [Ca2+] transient, we have opted not to implement the steps of the multistate model that deal with the Ca2+ unbinding from the SERCA on the SR luminal side of the pump. In brief, the reactions SERCA2a undergo are: (1) binding/unbinding of the first Ca2+; (2) binding/unbinding of the second Ca2+.To implement an approximated SERCA pump action, we used a simplified version of a multi-state kinetic model developed earlier2+ diffusivity was obtained from studies, in which it was concluded that given the known kinetics of typical Ca2+ buffers, the range of free Ca2+ after it enters a cell can be up to 200 nm. Therefore, due to the nano-scale of our system, the trajectories of Ca2+ released by TPC2 signaling complexes in the simulations are governed by the measured diffusivity of free Ca2+ for a displacement comparable to the nanojunction extension . Once Ca2+ are buffered they acquire the measured diffusivity of the buffers .The CaWe summarize the quantitative model input data in2+ release that is supposed to occur at L-SR junctions in PASM experiments. Released Ca2+ is assumed to undergo Brownian motion in the surrounding space, including the L-SR nanospace. SERCA2a placed on the neighbouring SR surface may capture Ca2+ according to our approximation of their known multistate model. To determine how this regenerated cellular environment can shape a Ca2+ transient we “measured” the junctional [Ca2+] by counting the ions within the L-SR volume at any given time and dividing the number by that volume. The snapshots in2+ transients in the L-SR junctions were collected in a measuring volume placed between the lysosomal and SR membranes in the virtual L-SR junction  over which maximum SR Ca2+ release via RyR3 is reported to occur in skeletal muscle58. This comparison underscores the important constraint played by the width of the L-SR junctions and indicates that, unless the membrane separation is kept below about 30 nm, it is not possible for [Ca2+]NJ to breach the threshold for RyR3 Ca2+ release. Let us also point out that the [Ca2+]NJ data int′transient <ttransient around the [Ca2+]NJ peak. However, in that caseNNJ  would have to contribute to the signal summation that results in a 5-secondtshoulder. Interestingly, this possibility agrees with our lysosome count from TEM images (110 lysosomes/cell) and with the argument of natural redundancy we contemplated to explain the discrepancy between the calculated and observed lysosome number estimates. As an exercise we have recalculated the [Ca2+]NJ time-averaged overt′transient obtained using 110 lysosomes in2+ release threshold would be cleared more readily. These observations suggest that activation of RyR3s in the L-SR junctions not only depends on the concentration of Ca2+ near them, but also on their exposure time to this concentration.To gain quantitative insight into the effects of varying the junctional width, we then calculated the temporal average of the [CaFigure_1_ms_suppl.csv Calcium fluorescence Fura-2-AM calcium fluorescence imaging data set used to produce the graphs in Figure 1 of the manuscript as well as Figure 1 in the supplement file to the main manuscript. Data are in a csv file with columns headers for indicating the data in each column.Figure_2_B-D.csv L-SR characterization Data for image analysis statistics shown in Figure 2B-D and gathered from transmission electron micrographs. Data are in comma-separated columns in a simple text file. Column headers indicate the quantity and units listed in each column.Figure_6A.txt TPC2 open probability vs lysosomal luminal [Ca2+] Data used for plot in Figure 6A and obtained from analysis of experimental results in Pitt S et al 2010 (see manuscript references). Data are in space/tab-separated columns in a simple text file. Column headers indicate the quantity and units listed in each column.Figure_6B.txt TPC2 Ca2+ release rate vs time Data used for plot in Figure 6B and obtained as described in section “L-SR junctional Ca2+ signal”. Data are in space/tab-separated columns in a simple text file. Column headers indicate the quantity and units listed in each column.Figure_7A_20_nm.txt, Figure_7A_50_nm.txt, Figure_7A_100_nm.txt Nanojunctional [Ca2+] vs time Data for plot in Figure 7A and obtained as average over 100 simulations each started with a different random number generator seed . The files contain data from simulations with L-SR junctional separations of 20, 50 and 100 nm, respectively. Data are in space/tab-separated columns in a simple text file. Column headers indicate the quantity and units listed in each column.Figure_7B.txt Nanojunctional [Ca2+] vs L-SR separation distance Data for plot in Figure 7B, solid circles, obtained by temporal average of data in figure 7A over entire time interval. Data are in space/tab-separated columns in a simple text file. Column headers indicate the quantity and units listed in each column.Figure_7B_short_t_avg.txt Nanojunctional [Ca2+] vs L-SR separation distance Data for plot in Figure 7B, empty circles, obtained by temporal average of data in figure 7A over a shorter time interval, as described in section “Reconciling the temporal scales in simulation and experimental results”. Data are in space/tab-separated columns in a simple text file. Column headers indicate the quantity and units listed in each column.Click here for additional data file.2+ release and uptake at a family of specific nanojunctions connected by a continuous but variable SR lumen integrates cellular control over multiple functions4. The lysosome-sarco/endoplasmic reticulum (L-S/ER) junction is the most current junction to be considered in this context and exhibits perhaps the highest degree of plasticity of the family of nanojunctions of the SR; the mechanism and function of lysosomal Ca2+ signaling is currently hotly debated59.We have recently introduced the concept of the “panjunctional SR”, which states that Ca2+ signals and optical immunocytochemistry11. Our observations corroborate the previously reported finding17 that lysosomes in PASMCs tend to cluster in the perinuclear region, as is evident in our micrographs  and extend approximately 300 nm in lateral dimensions, thereby involving about 15% of the lysosomal membrane . In the first instance, we have estimated the potential contribution of local bursts of Ca2+ release from individual lysosomes to the elevation of global [Ca2+]i observed during this shoulder in the experimental records. From this, and using the dimensions of a typical smooth muscle cell, we calculated that 60–100 lysosomes would be required to cause an elevation of comparable magnitude to the signal shoulder. This is lower than the estimate for the total number of lysosomes per cell, 110, we obtained from our ultrastructural study, but comparable in order of magnitude. We have already mentioned above a number of factors in favour of observing a greater number of lysosomes/cell than the estimated number required to generate the signal shoulder. In addition, several other elements contribute a degree of uncertainty to those estimates, such that their discrepancy may not be as large. We need to consider that publishedPo data for the Ca2+ conductance associated with the TPC2 signaling complex45, on which we based our TPC2 rate table, show some variability according to the standard deviation bars, which, in turn, implies an uncertainty in the interpolatedPo (NPo) rather than a purely single channelPo. Lastly, the standard deviation of the simulated transient , it is worth noticing that the values we find are comparable to those recently measured in so-called Ca2+ hot spots in the mitochondria-ER junctions of neonatal ventricular cardiomyocytes (rat culture)62 and in RBL-2H3 and H9c2 cells earlier63. These results therefore suggest that the hypothesis presented for the role of L-SR junctions in cellular Ca2+ signaling is certainly plausible.To take this study further and understand whether the observed L-SR junctions could give rise to shoulder. Remarkably, this value is comparable to the average duration of the simulated transients, which was determined on the basis of ultrastructural details and completely independently of the summation effect hypothesis. The assumption of summation may imply that the role of the NAADP as a stimulus is limited to the initial lysosomal Ca2+ release, while the recruitment of subsequent L-SR junctions may be governed by further lysosomal calcium release, by SR Ca2+ release via Ca2+ activated junctional RyR3, and/or propagation and combination of calcium signals via inter-junctional clusters of RyR3. Therefore, from our model we envision the intriguing possibility of an important regulatory role of lysosomal Ca2+ content by SR Ca2+, in such a way that summation of calcium signals at multiple junctional complexes may give rise to the shoulder. While only further experiments can verify this, the quantitative corroboration provided by our findings sets on firmer ground the conclusion from earlier studies that Ca2+ bursts from lysosomes are indeed responsible for initiating the first phase of a cell wide Ca2+ wave via L-SR junctions.In this respect, our results also allowed us to establish a time interval for the duration of the transient  An increase in junctional distance such that [Ca2+]NJ is insufficient to breach the threshold for activation of RyR2, yet sufficient to allow for increases in luminal Ca2+ load of the SR via apposing SERCA2 clusters; or (2) L-SR junctions in cardiac muscle formed between lysosome membranes and closely apposed regions of the SR which possess dense SERCA2 clusters and are devoid of RyR2. Further ultra-structural studies on cardiac muscle and other cell types may therefore provide a greater understanding of how L-SR junctions may have evolved to provide for cell-specific modalities within the calcium signaling machinery.Carrying the signal time-scale analysis further and using the calculatede RyR3s . Thus it65. It may be significant, therefore, that TPC2 gating and thus autophagy may be modulated by mTOR66. This may well speak to further roles for lysosomal calcium signaling and L-SR junctional plasticity during hypoxic pulmonary vasoconstriction and even during the development of hypoxia pulmonary hypertension67, not least because AMP-activated protein kinase has been shown to modulate autophagy through the phosphorylation and inhibition of mTOR68.Under metabolic stresses, such as hypoxia, lysosomal pathways have been proposed to provide for autophagic glycogen metabolism via acid maltase in support of energy supply, and significant levels of protein breakdown during more prolonged metabolic stress. Moreover, although controversial, in some cell types it has also been suggested that lysosomes may contribute to the energy supply by providing free fatty acids for beta-oxidation by mitochondria69. For this to occur L-SR junctions would likely be disrupted and thus select for local rather than global Ca2+ signals in order to facilitate fusion events among lysosomes, endosomes, autophagosomes and amphisomes70, which follows from the fact that Ca2+ plays a pivotal role in vesicle trafficking and fusion71. Active non-synchronous movements of TPC2-expressing vesicles have been detected in live-cell imaging experiments using GFP-tagged proteins20. Thus, lysosomal Ca2+ signaling may modulate plasticity in the manner required at all stages of multiple membrane fusion events that are dependent on Ca2+ for the effective formation of the SNARE complexes71. In short, transport of proteins between lysosomes, Golgi apparatus, and plasma membrane via lysosomes and endosomes may be coordinated at different stages by both global Ca2+ signals and spatially restricted Ca2+ release from acidic stores in a manner that is in some way determined by L-SR junctional integrity.The process of autophagy serves not only to regulate programmed cell death, but also to recycle organelles, such as mitochondria, through a process of degradation involving lysosomal hydrolases2+ release both depletes luminal Ca2+ and causes intraluminal alkalinization72. Therefore, disruption of L-SR junctions may modulate the activity of pH-sensitive hydrolytic lysosomal enzymes, such as glucocerebrosidase and acid sphingomyelinase, which exhibit a marked loss of function at pH > 573 that could lead to accumulation of macromolecules such as glucocerebroside and sphigomylin. As mentioned previously, dysfunction of these enzyme systems consequent to L-SR junctional abnormalities could also contribute to pathologies associated with subclasses of lysosome storage disease such as Niemann-Pick disease type C123, Pompe and Gaucher25, which may include hepatic portal23, or pulmonary hypertension25, dysfunctions in cholesterol trafficking26 and consequent increases in plasma cholesterol levels, vascular lesion formation, atherosclerosis/thrombosis and medial degradation27.Loss of integrity of L-SR junctions may also contribute to disease, given that lysosomal Ca2+ signals and strong support for the proposal that these Ca2+ signals are generated at L-SR junctions. Even within the variability of our recorded values of L-SR junctional widths and extension, our results suggest that localized [Ca2+] transients due to junctional Ca2+ release can without fail reach values required to breach the threshold for CICR from junctional RyR3s. Perhaps most significantly, disruption of the nanojunctions decreases [Ca2+]NJ below the value for CICR via junctional RyR3s. Therefore, consistent with previous studies on the PM-SR membrane39, we have established that L-SR junctions are required to allow such signals to be generated and that there is a 30–50 nm limit on junctional width, above which there is loss of junctional integrity and inadequate control of ion movements within the junctional space. This suggests that the observed L-SR junctions in PAMSCs are not only capable of delivering localized Ca2+ bursts of the required magnitude, but are also necessary if lysosomes are to fulfill this identified role in Ca2+ signaling. In other words, L-SR nanojunctions are a necessary and sufficient condition for generating local Ca2+ bursts essential for NAADP-induced Ca2+ waves. Most importantly, however, this study demonstrates the importance of junctional architecture on the nanoscale to the capacity for coupling across cytoplasmic nanospaces, tight regulation of ion transport and thus signal transduction. In turn, this highlights the possibility that alterations in the dimensions and architecture of intracellular nanojunctions lead to cell dysfunction and hence disease.We have determined that L-SR junctions, about 15 nm in width and extending to ≈ 300 nm in lateral dimensions are a regularly occurring feature in rat pulmonary arterial smooth muscle cells, in which L-SR junctions were first proposed. The present study provides a mechanistic basis for the observed NAADP-induced Ca10.6084/m9.figshare.988699figshare: L-SR nanojunction study data, doi: 2+ release responses were observed in smooth muscle cells, including pulmonary artery smooth muscle cells (PASMCs). Lysosomes and sarcoplasmic reticulum (L-SR) junctions had been proposed as the structural bases in facilitating local lysosomal Ca2+ bursts (first phase) followed by whole cell Ca2+ release responses from SR (second phase) through a calcium-induced calcium release (CICR) mechanism. In their studies reported here, Nicola Fameliet al. attempted to define the L-SR junctions in PASMCs with a nano-level resolution using electron microscopy (EM), and proceeded to build a quantitative L-SR junctional model. Unfortunately, all these efforts will add little to our further understanding of these L-SR junctional structures, if not at all, due to the lack of solid experimental EM results in reconstructing L-SR junctions, some dogmatic presumptions in L-SR model build up, the inadequacy of validation of the model and a failure to include information on the current disputes over the identity of NAADP-associated Ca2+ release channels in lysosomes. Some major concerns are as follows:NAADP mediated two-phase Ca12. However, such a standard practice was excluded in this study as a ‘precaution’ to prevent compromising lysosome membrane definition. In line with applying empirical judgments for identifying organelles to the EM data analysis, the authors extrapolated the opposing and proximal organelles adjacent to the nucleus as lysosomes and SR, and deemed them to be worth studying . In addition, the authors stated that there were few lysosomes found in the subplasmalemmal region, which implied that L-SR junctions were profoundly located around the nucleus. However, the fura-2 fluorescence images in Figure 1 as well as in the two citations used as basis of this study 34 exclusively demonstrated that the first phase lysosomal Ca2+ release initiated from a region around the cell membrane, suggesting that L-SR junctions should have cytoplasmic locations near the plasma membrane. It is clear that more reliable methods such as immune-gold staining are needed to resolve the contradictions over their own results.Data are not convincing and lack quality. In defining L-SR junctions, the assurance of lysosome and SR identities is fundamental. Immune-gold staining has been widely applied in EM studies to ascertain different cell organelles as well as to conduct 3D reconstruction of cell structure-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy”. In defining the L-SR nanojunctions, they declared that lysosomes and SR “must be separated by a distance of approximately 20 nm and be of the order of a few hundred nm in lateral dimensions” by citing PM-SR junctions studies 34. However, these two citations originated from the light microscope-based studies of L-SR junctions by the same group. Given the resolution limitation, these light microscopy results have no way to give an estimation as accurate as 20 nm. It is therefore confusing to find that these references fail to back up such claims, in my opinion. In reference 395, it has been proposed that the extracellular Ca2+ entered the cell through Na+/ Ca2+ exchanges, traversed PM-SR junctions to refill SR via Ca2+-ATPase and sustained cell Ca2+ oscillations. For the current L-SR model, this PM-SR modeling may be used as a comparison or reference system leading to the conclusion “that L-SR had a similar 20 nm gap measured as that in PM-SR junctions.” Given that there are no similarities of PM-SR to L-SR junctions, in terms of potency of Ca2+ movement due to Ca2+ gradient across PM and lysosomal membrane, biophysical property of Na+/ Ca2+ exchangers and TPC2 channels, and the actions of Ca2+-ATPase and RyR3 Ca2+ receptors, such a conclusion is not reliable from the modeling. In addition, a major concern is that this model is too simplistic as it excludes many other Ca2+ regulatory mechanisms in both lysosomes and SR, which may influence the accuracy of junction width or prediction in modeling.The modeling is based on many assumed parameters and lacks reliable validation. To establish the L-SR nanojunctional model, the present study has introduced many assumptions to some critical parameters such as the distance between the lysosome and SR, the size of lysosome, and total number of lysosomes; many of which lack proper judgments. In the abstract section, the authors rationalized the application of EM in investigating L-SR junctions by stating “L2+ signal , the authors introduced up to four assumptions and calculated that ~60-100 lysosomes were needed to generate this shoulder peak. They verified these lysosome numbers with their TEM images that “can expect a total of about 110 lysosomes/cell”. Considering these two calculations however, it is obviously problematic because almost all lysosomes (100 out of 110 each cell) will need to functionally contribute to the L-SR junctional structure formation. In addition, TEM measurements showed the lysosome dimensions (Table 1) to be major axis of 398 ± 91 nm and minor axis of 325 ± 65 nm. Given the L-SR junction model features “junctional membrane separation and extension are about 15 nm and 300 nm”, it could be postulated that all the lysosomes that participated in L-SR structure formation also have to align well with the SR in order to produce a 300 nm junctional extension. Therefore, this L-SR model actually describes a scenario whereby in PASMCs the majority of lysosomes act as the components of L-SR junctions and uniformly have impeccable arrangements to oppose the SR at 20 nm apart. However, this idealistic model largely contradicts the well-established concepts that lysosomes are active organelles that dynamically exert their different functions by their movements and other activities.Some assumptions contradict well-established concepts due to a lack of consideration of lysosome biology. In explaining the shoulder Ca2+ release channels are questionable. In quantifying the L-SR model, the authors arbitrarily assumed TPC2 as the only lysosomal NADDP-activated Ca2+ channel by totally disregarding recent findings that TPC2 is not a lysosomal NAADP-associated Ca2+ channel, but a Na+ channel6,7 . They also ignored the contribution of other lysosomal channels or transporters that may contribute to such L-SR junction activity. This prejudice in channel selection inevitably invites doubts on the reliability of this proposed L-SR model. In addition, the authors collectively adopted electrophysiological data from other research groups without taking into consideration any incompatibilities with the model. For example, in obtaining the total number of TPC2 channels on a lysosome, the authors2+ current in isolated lysosomes (48)8, by the single channel conductances(45)9, thus estimating that a typical lysosomal membrane may contain ~20 TPC2“…dividing the macroscopic whole lysosome conductance, calculated from the current values reported in a recent study on TPC2-mediated Ca”. Unfortunately, the lysosomes used in the whole lysosome conductance recording were not normal lysosomes, but lysosomes manipulated by overexpression of TPC2 and were undergoing size enlargement. With these changes, the modified lysosomes would have a much higher TPC2 conductance compared with normal lysosomes with a natural biogenesis. It is important to use a TPC2 conductance and other parameters of channels from lysosomes of normal cells without an artificial transgene for such modeling. Otherwise, the modeling prediction will be inaccurate and the relevance of such a model will be vague.TPCs as lysosomal Ca            In conclusion; aside from the issue that the current L-SR nanojunction model fails to contribute any new insights to our understanding of the L-SR architecture, my concerns over the reliability of data and modelling parameters are rather serious – this work is therefore ‘Not Approved’ in its current version.I have read this submission. I believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. 1) would be particularly advantageous.This is certainly an interesting study. The characterisation of lysosomal-SR junctions is novel and conceptually important. The data look convincing but I would recommend the authors to further expand the structural part of the study. It would perhaps be useful to further verify that the organelles participating in the formation of the contacts are indeed lysosomes and that the junctions are not simply the result of accidental collisions of the two organelles. Identifying/visualising tethers between the organelles . One could also perhaps consider measuring this important parameter using isolated lysosomes.Modelling part of the paper is also useful. In this respect it would be valuable to clarify the CaI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. 2+ transients; these are produced by release of Ca2+ from lysosome stores that induce Ca2+-induced Ca2+ release from SR stores via stimulation of RyR3. It is increasingly apparent that answers to many issues concerning cellular Ca2+ signalling will be found by recording local Ca2+ events – for instance the recently identified Ca2+ sparklets representing Ca2+ influx through opening of plasma membrane ion channels.This is an interesting and thought provoking study which simulates NAADP-dependent Ca2+ events might be modeled between discrete organelles. Moreover, unlike many simulation manuscripts, I find this one clear and well-written.  Simulation studies are always difficult to review and evaluate as they must make many assumptions. However, this work is considered and may be a useful model for how other local Ca2+ following NAADP/TPC2 activation – a role for a lysosome store-operated Ca2+ influx?Do the authors think that the use of proximity ligation assay techniques might be appropriate to measure local interactions between TPC2 and RyR3? Also, how are lysosomes refilled with CaI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Here we report on specific anion effects on the ATP-dependent Ca2+ translocation by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Current measurements following ATP concentration jumps on SERCA-containing vesicles adsorbed on solid supported membranes were carried out in the presence of different potassium salts. We found that monovalent anions strongly interfere with ATP-induced Ca2+ translocation by SERCA, according to their increasing chaotropicity in the Hofmeister series. On the contrary, a significant increase in Ca2+ translocation was observed in the presence of sulphate. We suggest that the anions can affect the conformational transition between the phosphorylated intermediates E1P and E2P of the SERCA cycle. In particular, the stabilization of the E1P conformation by chaotropic anions seems to be related to their adsorption at the enzyme/water and/or at the membrane/water interface, while the more kosmotropic species affect SERCA conformation and functionality by modifying the hydration layers of the enzyme.The occurrence of Hofmeister (specific ion) effects in various membrane-related physiological processes is well documented. For example the effect of anions on the transport activity of the ion pump Na They occur in simple bulk solutions and at interfaces, in water and in non-aqueous solutions, and consist in the different effect that salts exert in a particular system1The occurrence of specific ion effects in biochemistry is very well documented by an enormous number of papers110132+-ATPase (SERCA). SERCA is a well characterized cation transport ATPase1617182+ ions from the cytoplasm to the SR lumen, thus inducing muscle relaxation. It is now well established that the Ca2+ pump also countertransports two to three protons and that the transport cycle is electrogenic202122The aim of the present research is to investigate specific ion effects on the pumping activity of the sarcoplasmic reticulum Ca1-E2 scheme (2+ per enzyme molecule (E1Ca2), followed by phosphorylation by ATP and formation of a phosphoenzyme intermediate (E1P). The free energy derived from ATP is used by the phosphoenzyme for a conformational transition (E1P to E2P) that favours the translocation and release of the bound Ca2+ against its concentration gradient. Ca2+ ions are delivered to the intravesicular lumen in exchange for lumenal protons, that are translocated across the membrane to the cytosolic side during the following enzyme dephosphorylation. Hydrolytic cleavage of the phosphoenzyme is the final step, which allows the enzyme to undergo a new transport cycle.According to the E2 scheme  derived +, K+-ATPase. The interaction of anions with Na+, K+-ATPase was characterized by biochemical and equilibrium fluorescence measurements, kinetic experiments using fluorescence dyes and electrical measurements on bilayer lipid membranes252627282930+, K+-ATPase kinetics and transport activity follows the Hofmeister series. On the other hand, only a few studies on the interaction of anions with SERCA were reported. It was shown that certain anions can affect Ca2+ and H+ gradients produced by SERCA in reconstituted proteoliposomesIt is known that various anions are able to affect partial and overall enzymatic reactions of the electrogenic ion pump Na2+ translocation by SERCA, we employed an electrical method, which makes use of a solid supported membrane (SSM), depicted in 343536373839e.g. Ca2+ or ATPi.e. Ca2+ ions, within the protein35To investigate the effects of several anions on the ATP-dependent Ca2+ translocation, and discuss the results in terms of the conformational transition between the phosphorylated intermediates E1P and E2P of the SERCA cycle.In this contribution we report on the current measurements following ATP concentration jumps on SERCA-containing vesicles adsorbed on SSMs, in the presence of different potassium salts. We detected a specific ion effect on the ATP-induced Ca2+ translocation by SERCA, keeping the cation (K+) constant. The effect of the different anions was compared to that of chloride, which represents the reference anion.We performed current measurements on native SR vesicles containing SERCA adsorbed on a SSM, in order to investigate the effect of different potassium salts on the ATP-dependent Ca2+, 1 mM Mg2+ and 100 mM potassium salt at pH 7. For each selected anion, the corresponding ATP-induced current transient was compared to that measured in the presence of 100 mM KCl, taken as a control measurement , whereas a slight increase in the translocated charge was observed in the presence of 300 mM chloride42− anion (100 mM K2SO4) we observed a significant increase in the current amplitude (and related charge) with respect to the current detected in the presence of 100 mM KCl , thereby confirming that the increase in current amplitude and associated charge in the presence of sulphate can be ascribed to the specific nature of the SO42− anion and not to the different ionic strength of the solution.We recall here that in the case of Na0 mM KCl . In the norm and some of the most important ionic physico-chemical parameters, that account for ion specificity.In order to investigate the mechanism through which the different anions affect the translocated charge in a specific manner, we checked the correlation between the values of Qnorm as a function of the anion static polarizability (α), of the surface tension molar increment (σ) and of the hydration entropy (ΔShydr), respectively. These parameters reflect the different nature of the ions. The former represents the softness of the electron cloud and contributes in setting the extent of the frequency-dependent dispersion forces (see eq. 14 in ref. e.g. below 10−3 M), non-electrostatic forces emerge when the concentration of the ions is rather large  and directly depend on the nature of the single ionnorm with the air/water surface tension molar increment and with the lysozyme/water surface tension molar increment, respectively. This parameter is defined as σ = ∂Δγ/∂c, and measures the effect of each ion on the air/water surface tension change (Δγ) as a function of the salt concentration (c)LW, i.e. the surface tension molar increment at the lysozyme/water interfacenorm versus σLW. In fact this protein/water interface is certainly closer to the system studied in the present paper. In any case, the trends shown in norm, as a result of the ion adsorption at the membrane/water or at the enzyme/water interface, and therefore suggests that the specific interfacial adsorption of the ions is involved in the mechanism that modifies the pumping activity of SERCA, depending on the nature of the background electrolyte.One may argue that in our study ions do not interact with an air/water interface, but rather with a membrane/water or with an enzyme/water interface. For this reason we listed in norm as a function of the ion hydration entropy. ΔShydr reflects the ability of an ion to polarize the water molecules that reside in the first hydration shell around the enzyme and/or the membrane2+ ions to SERCA was carried out without ATP. In particular, 10 μM free Ca2+ concentration jumps were performed and the corresponding current signals were recorded. The charge obtained by numerical integration of the Ca2+-induced current transient is due to the electrogenic event related to binding of Ca2+ ions to SERCA from the cytoplasmic side in the absence of ATP35363−) and for a typical kosmotropic (SO42−) anion, at a concentration of 100 mM, are 0.86 ± 0.02 and 0.97 ± 0.11, respectively. These charges are normalized to the value obtained in the presence of 100 mM KCl (control measurement).Before proceeding with the discussion of the charge translocation data, we point out that no specific ion effect was detected when the binding of Ca2+ to the ATPase transport sites, i.e. the E1 → E1·Ca2 step in the diagram depicted in The results indicate, within the experimental error, that the displaced charge is not significantly affected by the presence of 100 mM nitrate or sulphate, thereby suggesting that the two anions do not interfere with the binding of cytoplasmic Ca2+ translocation by SERCA in terms of the conformational transition between the phosphorylated intermediates E1P and E2P of SERCA, i.e. the E1P → E2P transition -containing vesicles adsorbed on solid supported membranes (SSM), in the presence of different potassium salts. We detected a significant specific ion effect on the ATP-induced calcium translocation, and discuss the results in terms of the specific adsorption and of the hydration properties of the anions at the interface between the enzyme or the vesicle and the aqueous surrounding medium.We report on the current measurements following ATP concentration jumps on sarcoplasmic reticulum Ca1P and E2P) and modify the performance of the enzyme. For the chaotropes the main effect is due to their adsorption at the enzyme/water interface, while the more kosmotropic species (sulfate and chloride) act in perturbing the hydration layers of the enzyme and therefore in modifying its conformation and functionality.These results seem to indicate that the effect of the anions is related to their ability to stabilize either of the two phosphorylated intermediates . The SSM consists of an octadecanethiol monolayer covalently bound to the gold surface via the sulphur atom, with a diphytanoylphosphatidylcholine monolayer on top of it, as illustrated in Fig. 234e.g. Ca2+ or ATP. If at least one electrogenic step, i.e. a net charge movement across the vesicular membrane generated by the protein is involved in the relaxation process that follows the protein activation, then a current transient will be detected due to the capacitive coupling between the vesicle membrane and the SSM3536i.e. at zero applied voltage relative to the reference electrode. The numerically integrated current transient is related to a net charge movement within the protein, which depends upon the particular electrogenic step (i.e. following Ca2+ or ATP concentration jumps).SR vesicles, following a brief sonication, were adsorbed on the SSM surface during an incubation time of 60 min. After the adsorption, SERCA was activated by the rapid injection of a solution containing an appropriate substrate, 2+ concentration-jump experiments, the non-activating solution contained 100 mM potassium salt, 10 mM 3-(N-morpholino)propansulfonic acid , 0.25 mM ethylene glycol tetraacetic acid (EGTA) and 1 mM MgCl2; the activating solution contained, in addition, 0.25 mM CaCl2 (10 μM free Ca2+).In Ca2, 0.25 mM EGTA and 0.25 mM CaCl2 (10 μM free Ca2+); the activating solution contained, in addition, 100 μM ATP.In ATP concentration-jump experiments, the non-activating solution contained 100 mM potassium salt, 10 mM MOPS (pH 7.0), 1 mM MgClThe effect of potassium salts on charge movements was investigated at a salt concentration of 100 mM, keeping constant the concentrations of the other solutes. In all experiments two buffered solutions were employed, the “non-activating” and the “activating” solution:2+ concentration was calculated with the computer program WinMAXCFree Ca2ROne device (Nanion Technologies). The temperature was maintained at 22–23 °C for all the experiments.The concentration jump experiments were performed by the SURFETo verify the reproducibility of the current transients on the same SSM, each single measurement was repeated 6 times and then averaged to improve the signal to noise ratio. Standard deviations did not exceed 5%. Moreover, each set of measurements was usually reproduced using two-three different SSM sensors.How to cite this article: Tadini-Buoninsegni, F. et al. Hofmeister effect of anions on calcium translocation by sarcoplasmic reticulum Ca2+-ATPase. Sci. Rep.5, 14282; doi: 10.1038/srep14282 (2015).


Unlike other N-terminal disease-associated mutations, the H29D mutation is located on the surface of the N-terminal domain. It is unclear how this surface-exposed H29D mutation that does not appear to interact with other parts of the RyR2 structure could alter the intrinsic properties of the channel. Here we carried out detailed functional characterization of the RyR2-H29D mutant at the molecular and cellular levels. We found that the H29D mutation has no effect on the basal level or the Ca2+ dependent activation of [3H]ryanodine binding to RyR2, the cytosolic Ca2+ activation of single RyR2 channels, or the cytosolic Ca2+- or caffeine-induced Ca2+ release in HEK293 cells. In addition, the H29D mutation does not alter the propensity for spontaneous Ca2+ release or the thresholds for Ca2+ release activation or termination. Furthermore, the H29D mutation does not have significant impact on the thermal stability of the N-terminal region (residues 1–547) of RyR2. Collectively, our data show that the H29D mutation exerts little or no effect on the function of RyR2 or on the folding stability of the N-terminal region. Thus, our results provide no evidence that the H29D mutation enhances the cytosolic Ca2+ activation of RyR2.The N-terminal domain of the cardiac ryanodine receptor (RyR2) harbors a large number of naturally occurring mutations that are associated with stress-induced ventricular tachyarrhythmia and sudden death. Nearly all these disease-associated N-terminal mutations are located at domain interfaces or buried within domains. Mutations at these locations would alter domain-domain interactions or the stability/folding of domains. Recently, a novel RyR2 mutation H29D associated with ventricular arrhythmia at rest was found to enhance the activation of single RyR2 channels by diastolic levels of cytosolic Ca The cardiac ryanodine receptor RyR2) is an essential component of excitation-contraction coupling in the heart ,2. RyR2  is an esRecent major advances in defining the three-dimensional (3D) structure of RyR have provided invaluable and detailed insights into the molecular mechanisms of disease-associated RyR mutations. For instance, high-resolution structures of the N-terminal region of RyR have recently been solved –16. The 2+ activation at diastolic cytosolic Ca2+ concentrations compared to single RyR2 WT channels ryanodine binding to RyR2. Single channel analysis showed that the H29D mutation did not affect cytosolic Ca2+ activation of single RyR2 channels. The H29D mutation also had no effect on the activation of RyR2 by caffeine or the propensity for store-overload induced Ca2+ release (SOICR). The H29D mutation also did not alter the cytosolic Ca2+ dependence of Ca2+ release or the activation or termination threshold for SOICR. Furthermore, the H29D mutation had no effect on the expression of the RyR2 channel or the thermal stability of the N-terminal domains of RyR2. Collectively, our data indicate that the H29D mutation does not significantly alter the intrinsic properties of RyR2. Our results do not support the notion that the H29D mutation causes a 'leaky' RyR2 channel.A novel RyR2 mutation, H29D, has recently been identified and is believed to be associated with short-coupled polymorphic ventricular tachycardia at rest . Functiochannels . It was  at rest . However at rest ,12,13, t at rest –16. MorePoint mutation, H29D, in the mouse RyR2 was generated by the overlap extension method using PCR ,25. Brie2+ phosphate precipitation method. The transfected cells were washed with PBS 24 h after transfection followed by a change into fresh media for 24 h. The cells were then washed again with PBS, harvested, and plated onto new dishes. After the cells had attached (~4 h), the growth medium was replaced with a selection medium containing 200 μg/ml hygromycin (Invitrogen). The selection medium was changed every 3–4 days until the desired number of cells was grown. The hygromycin-resistant cells were pooled, aliquoted, and stored at -80°C. These positive cells are believed to be isogenic, because the integration of RyR2 cDNA is mediated by the Flp recombinase at a single FRT site. The handling and storage of the human embryonic kidney 293 (HEK293) cell line were approved by the Institutional Biosafety Committee at the University of Calgary.Stable, inducible HEK293 cell lines expressing RyR2-WT or the RyR2-H29D mutant were generated using the Flp-In T-REx Core Kit from Invitrogen . Briefly3H]ryanodine binding to cell lysates was performed as described previously [3H]Ryanodine binding was carried out in a total volume of 300 μl binding solution containing 30 μl of cell lysate, 100 mM KCl, 25 mM Tris, 50 mM Hepes (pH 7.4), 5 nM [3H]ryanodine and CaCl2 to set free [Ca2+] from pCa 9.89 to pCa 4 and a protease inhibitor mix at 37°C for 20 min. Ca2+/EGTA ratio was calculated using the computer program of Fabiato and Fabiato [3H]ryanodine binding in the presence of 50 μM unlabeled ryanodine. All binding assays were done in duplicate.HEK293 cells were plated in 100 mm tissue culture dishes at ~10% confluence 18~20 h before transfection with RyR2 WT or H29D mutant cDNA. After transfection for 24 h, the cells were harvested and lysed in lysis buffer containing 25 mM Tris, 50 mM HEPES, pH 7.4, 137 mM NaCl, 1% CHAPS, 0.5% egg phosphatidylcholine, 2.5 mM DTT, and a protease inhibitor mix  on ice for 60 min. Cell lysate was obtained after removing the unsolubilized materials by centrifugation twice in a microcentrifuge at 4°C for 30 min each. Equilibrium ryanodine binding has commonly been used as an index for the open probability (Po) of RyR. Furthermore, because [3H]ryanodine, once it is bound to RyR, dissociates from the channel very slowly, equilibrium [3H]ryanodine binding assay performed over several hours is highly sensitive to detect RyR openings especially under conditions where the Po of the channel is very low. Therefore, we employed [3H]ryanodine binding assay to determine the impact of the H29D mutation on the basal activity of RyR2 at low Ca2+ concentrations and the Ca2+ dependent activation of the channel. As shown in 3H]ryanodine binding to RyR2 at low Ca2+ concentrations (45–150 nM) (50 of Ca2+ dependent activation of [3H]ryanodine binding between the RyR2 WT (0.193 ± 0.002 μM) and H29D mutant (0.203 ± 0.008 μM) (P = 0.338) (3H]ryanodine binding to lysate from HEK293 cells co-transfected with RyR2 WT and H29D (in 1:1 ratio) to mimic the heterozygous expression of H29D mutant in patients (i.e. H29D+/-). We found that H29D+/- does not affect the EC50 of Ca2+ dependent activation of [3H]ryanodine binding (0.200 ± 0.002 μM) (P = 0.914). It should be noted that the expression level of the RyR2 WT and H29D mutant are similar  . Further= 0.338) . We also similar . Thus, o2+ activation of RyR2, we determined the response of single RyR2 WT and the H29D mutant channels to various cytosolic Ca2+ concentrations (45nM to 50 μM) using single channel recordings in planar lipid bilayers. As shown in 2+ levels (2+ concentration tested (P = 0.148), and the mean closed time were 17.91 ± 7.20 ms for WT and 11.06 ± 3.20 for H29D (P = 0.409). Thus, the H29D mutation does not significantly affect the Po or the gating of the RyR2 channel at low cytosolic Ca2+ concentrations or the response of the channel to cytosolic Ca2+ activation.To directly assess the impact of the H29D mutation on the cytosolic Ca+ levels . There wn tested . Further2+ in the cellular context. We measured the steady state ER Ca2+ level in permeabilized HEK293 cells ryanodine binding, enhanced the propensity for spontaneous Ca2+ release, reduced the threshold for Ca2+ release activation and termination, and decreased the thermal stability of the N-terminal region of RyR2 ryanodine binding, the propensity for spontaneous Ca2+ release, the threshold for Ca2+ release activation or termination, or the thermal stability of the N-terminal region of RyR2. Thus, our data on the impact of both the G230C ryanodine binding and Ca2+ release assays, single cell Ca2+ imaging, and structural analysis clearly demonstrate that the H29D mutation has little or no impact on the intrinsic function of mouse RyR2 or the structural stability of the N-terminal region.Unlike the G230C mutation, Cheung et al. [ at rest ,12–16. H at rest , and is he G230C  and H29Dhe G230C  and Cheu2+ release by affecting protein-protein interactions that regulate RyR2 function. Further investigations will be needed to determine whether the RyR2-H29D mutation alters RyR2 function and SR Ca2+ release in the context of cardiac cells, intact heart or animal models.It is, however, unclear how the H29D mutation that does not alter the intrinsic function of the RyR2 channel causes ventricular tachyarrhythmia at rest. Although the H29D mutation does not affect the isolated RyR2 channel, it may alter the interactions between RyR2 and its regulatory proteins in cardiac cells. The H29 residue is exposed on the surface at the top of the cytosolic assembly of the RyR2 structure. Currently, no auxiliary protein has been shown to interact directly with the RyR N-terminal region, but the possibility remains that such a binding partner exists. This part of the RyR2 structure may interact with proteins associated with the transverse tubular membrane. Thus, it is possible that the RyR2-H29D mutation may alter excitation-contraction coupling or SR Ca2+ activation of RyR2, whether the H29D mutation has any effect on luminal Ca2+ activation of RyR2 has yet to be determined. We have previously shown that a number of CPVT RyR2 mutations enhance the activation of the channel by luminal Ca2+ and the propensity for spontaneous Ca2+ release upon store Ca2+ overload (SOICR) [2+ activation, the lack of effect of H29D on SOICR suggests that the H29D mutation is unlikely to exert a major impact on luminal Ca2+ activation. However, further studies are required to directly assess the impact of H29D on the luminal Ca2+ activation of RyR2.Although the H29D mutation has no effect on the cytosolic Ca (SOICR) ,20,26,312+ imaging techniques. Our data show that, in contrast to a previous study [2+ activation of RyR2. Our results also demonstrate that the H29D mutation does not alter the propensity for spontaneous Ca2+ release, a well-known cause for triggered arrhythmia.In summary, in the present study, we have characterized the impact of a recently identified RyR2 mutation H29D that is thought to be associated with ventricular arrhythmia at rest using a number of biochemical, electrophysiological, and Caus study , the RyR


Emerging optogenetic technology has forced the spectrally compatible GECI color variants. In this opinion, we highlight the recent development of GECIs including photo-switchable Ca2+ indicators and bioluminescence-based Ca2+ indicator, mainly invented in our group, focusing especially on the parameters determining their performance in order to provide a guideline for the selection of appropriate GECI for a given experiment.Fluorescent protein-based indicators are invaluable tools for functional imaging of living cells and organisms. Genetically encoded calcium indicators (GECIs) such as derivatives of yellow cameleons (YCs) and GCaMPs/pericams  at resting state and the amplitude of [Ca2+] change differ significantly within the subcellular locations, cell types, and organisms, a diverse set of affinity variants of GECIs covering dissociation constants (Kds) from nM to mM would be needed for studying a wide range of research targets.After the first reports regarding design concept of YCs and GCaMPs/pericams  were developed successfully by either site-directed mutagenesis of the Ca2+ binding domain in the indicator  and its binding peptide M13 had much higher Ca2+ affinity (Kd of 20 nM) than that of the CaM and M13 fusion protein linked with two amino acid linkers (Kd of 80 nM)  , which exist in abundance within the cell. Of course, excessive loading of Ca2+ indicator/chelator beyond the buffering capacity of these buffers does affect cellular Ca2+ homeostasis. In cases where more than mM concentrations of EGTA were loaded to observe subcellularly localized Ca2+, Ca2+ puffs and blips were generated , a skeletal and cardiac muscle-specific Ca2+-binding protein, is known to limit its interaction to just troponin I and troponin T. Indicators incorporating TnC from avian skeletal muscle or human cardiac muscle were generated, based on molecular design similar to that of cameleon. The resulting TN-L15 and TN-hTnC displayed a moderate Ca2+ affinity but a lower Ca2+ specificity (due to its cross reactivity with Mg2+) and a small signal change   can help overcome this limitation. Fluorescence status of PS-GECIs can be switched through light irradiation in arbitrary cell/tissue/organ, enabling cell/tissue/organ-specific visualization of Ca2+ dynamics. This “highlighted Ca2+ imaging” is beneficial in elucidating the activity of a single cell in the convoluted cell population of the neuronal network. There are only two reported PS-GECIs: a photoactivatable GECI, PA-TNXL  , a light-gated ion channel, and halorhodopsin (HR), a pump, allow us to control the activity of neural circuits with fine spatio-temporal resolution , the latest version of bioluminescence-based GECIs  was constructed by direct fusion of Venus with RLuc8, which is split by insertion of CaM-M13. Ca2+ binding to CaM-M13 induces reconstitution of the split Rluc8. This leads to FRET from reconstituted Rluc to Venus, resulting in a large increase in emission signal (2+ dynamics is monitored as a total intensity change, from both RLuc8 and FRET-enhanced emission of Venus. In the state-of-the-art demonstration by using cultured hippocampal neurons, Ca2+, transiently triggered by photo-activated ChR2, were imaged at 10 Hz with high SNR, showing the good compatibility of bioluminescence imaging with optogenetics (Saito et al., Nano-lantern (Ca2+ dyes in their targetability and reliability for chronic imaging. However, there remains room for further improvement on several parameters. Suboptimal kinetic property, non-linearity due to cooperativity, and pH sensitivity of single-FP based indicator should be improved to perform reliable detection of Ca2+. The future development of GECI is, regardless of the faults, promising, because of its evolvability. As a result of the past and the current attempts, not only have the basic property of GECIs been optimized, but also of new family of GECIs have been successfully developed, including color variants and self-illuminating GECIs. Future experiments will focus on improving the compatibility of GECIs with optogenetic tools. GECIs, used in collaboration with latest imaging platform such as a deep tissue imaging and ultra-fast and large-scale recording systems, will pave way to deepening our understanding of the supple ability our brain to learn and to memorize.GECIs are advantageous over synthetic CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TP53, the leukemogenic mechanism in the nearly 30% of CK-AML patients with wild-type TP53 has remained elusive. In this study, 15 cases with complex karyotype and wild-type TP53 were screened out of 140 de novo AML patients and the expression levels of MDM4, a main negative regulator of p53-signaling pathway, were detected. We ruled out mutations in genes associated with a poor prognosis of CK-AML, including RUNX1 or FLT3-ITD. The mRNA expression levels of the full-length of MDM4 (MDM4FL) and short isoform MDM4 (MDM4S) were elevated in CK-AML relative to normal karyotype AML (NK-AML) patients. We also explored the impact of MDM4 overexpression on the cell cycle, cell proliferation and the spindle checkpoint of HepG2 cells, which is a human cancer cell line with normal MDM4 and TP53 expression. The mitotic index and the expression of p21, BubR1 and Securin were all reduced following Nocodazole treatment. Moreover, karyotype analysis showed that MDM4 overexpression might lead to aneuploidy or polyploidy. These results suggest that MDM4 overexpression is related to CK-AML with wild-type TP53 and might play a pathogenic role by inhibiting p53-signal pathway.Acute myeloid leukemia patients with complex karyotype (CK-AML) account for approximately 10–15% of adult AML cases, and are often associated with a poor prognosis. Except for about 70% of CK-AML patients with biallelic inactivation of  TP53 mutations and have biallelic inactivation of TP53TP53 alterations.Acute myeloid leukemia patients with complex karyotype (CK-AML) account for approximately 10–15% of adult AML, and the incidence increases with age. CK-AML is characterized by chemoresistance, higher rates of refractory disease, and poor prognosis TP53 gene mutations. These reports suggest that overexpression MDM4 may be involved in the leukemogenic mechanisms of CK-AML patients without TP53 alterations. This question has not been fully explored to date.MDM4 is a negative regulator of p53, and by binding p53, close the transcriptional activity domain and thereby inhibits p53 function MDM4S and MDM4FL in CK-AML patients with wild-type TP53. We also measured cell proliferation, cell cycle, proteins related to p53 pathway and spindle checkpoint expression levels, and analyzed karyotypes in MDM4-overexpressing tumor cell line with wild-type TP53. We used these approaches to investigate the possible pathogenesis of MDM4-overexpression in CK-AML patients lacking TP53 mutations.In this study, we detected the expression levels of This study complies with the Declaration of Helsinki, and has been approved by the Ethics Committee of Shanxi Medical University. The written informed consent was obtained from all patients and from the legal guardians in the case of minors.de novo AML patients. The fusion genes RUNX11/RUNX1T1, PML/RARα or CBFβ/MYH11 of the patients were identified to be negative at the time of enrollment.Bone marrow samples were collected at the time of diagnosis of 140 non-M3 Conventional cytogenetics was performed at the time of diagnosis in 140 patients. Bone marrow cells were cultured in RPMI 1640 medium with 10% fetal bovine serum and penicillin-streptomycin for 24 hours, followed by treatment with 0.01 mg/ml colcemid for 60 min. Cells were harvested and placed in 0.075 M KCl for 15 min. After several changes in methanol-acetic acid fixative, slides were prepared by hot-plate drying. Metaphase chromosomes were banded by the trypsin-Giemsa or Phosphate R technique, and karyotyped according to the International System of Human Cytogenetic Nomenclature (ISCN 2005).TP53 gene and exon 3–9 of RUNX1 were amplified by PCR from genomic DNA and sequenced directly in all cases with complex karyotype. TP53 deletions were detected by interphase FISH in complex karyotype cases. Fms-related tyrosine kinase 3 length mutation (FLT3-ITD) analysis was performed as published TP53 and NK-AML patients.Exons 3–9 of the st Strand cDNA Synthesis Kit  and used in quantitative real-time PCR reactions with SYBR Premix Ex Taq (TaKaRa) and 0.5 µM of forward and reverse primers. For each gene analyzed, cDNA from 5×106 bone marrow cells of CK-AML or NK-AML patients were used for amplification. Primers used were as follows:For quantitative RT-PCR, cDNA was prepared using PrimeScript 15′-CAGCAGGTGCGCAAGGTGAA-3′MDM4FL-F: 5′-CTGTG CGAGA GCGAG AGTCTG-3′MDM4FL-R: 5′-CAGCAGGTGCGCAAGGTGAA-3′MDM4S-F: MDM4S-R: 5′-GCACTTTGCTGTAGTAGCAGTG-35′-GAGTTCATGACCTAC GGGAACCT-3′ABL-F: 5′-GGTACTCCATGGCTGACGAGAT-3′ABL-R: MDM4FL, MDM4S, and ABL, as well as the ΔCt (CtMDM4FL-CtABL or CtMDM4S-CtABL) was determined. NK-AML patients were set to 1 and relative expression graphed for MDM4FL and MDM4S mRNA in CK-AML patients. 2−ΔΔCt was used for calculating relative quantification.PCR conditions: initial denaturation at 95°C 10 s; denaturation 95°C 15 s, annealing 60°C 30 s, 40 cycles. The average Ct for HepG2 and 293T cell lines were obtained from the Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China. Cells were maintained in DMEM  supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 100 µg/ml streptomycin . Nocodazole (Sigma) was dissolved in DMSO and used at either 0.1 µg/ml or 1 µg/ml.EcoR I and BamH I respectively, and then linked with T4 DNA ligase (TakaRa). Plasmid sequences were confirmed by sequencing. Approximately 5×106 293T cells in 100 mm dishes was cotransfected with 10 µg pCDH1-MCS1-EF1-copGFP vector, pCDH1-MDM4FL-EF1-copGFP, or pCDH1-MDM4S-EF1-copGFP along with 10 µg packaging vector pPACKH1-GAG, pPACKH1-REV and pVSV-G using calcium phosphate precipitation. Media containing lentivirus were collected 48 and 72 hours after transfection and supernatant added to 5×105 HepG2 cells/well of a 6-well plate with 8 µg/ml polybrene (Sigma). For infection, cells were centrifuged at 1400×g for 2.5 hours at 32°C. GFP-positive cells were screened by limiting dilution, expanded in culture, and GFP-positive cells were pooled. To confirm that MDM4 was transfected into HepG2 cells, the expression levels of MDM4FL and MDM4S proteins were evaluated by western-blot analysis.For construction of the pCDH1-MDM4FL-EF1-copGFP and pCDH1-MDM4S-EF1-copGFP, MDM4FL or MDM4S fragments and pCDH1-MCS1-EF1-copGFP plasmid were digested by Cells stably expressing MDM4FL, MDM4S or vector control were cultured overnight and 0.1 µg/ml Nocodazole added the following day, and cells incubated for an additional 18 hours. Cells were stained by propidium iodine (PI) and cell cycle stage determined by flow cytometry (FCM). Cell proliferation was analyzed using the MTT assay. After 4 h incubation with MTT reagent, cells were lysed with DMSO for 10 min at 37°C and absorbance measured at 570 nm. The average percentage is shown for three independent HepG2 control, MDM4FL or MDM4S-expressing pools.6 control, MDM4FL or MDM4S-expressing cells, and stored at −80°C before use. Lysates (30 µg) were resolved by 8–12% SDS-PAGE and gels transferred to nitrocellulose membrane. Membranes were blocked with 5% nonfat milk in PBST for 1 h followed by primary antibody and incubation overnight at 4°C with gentle rotation. Membranes were washed twice with PBS containing 0.2% Tween 20 and incubated with appropriate secondary antibodies for 1 h at room temperature with gentle rotation. Membranes were then washed twice with PBST and incubated with Super Signal West pico  for 1 minute and exposed to film. Images were captured using the Bio-Rad ChemiDoc Imager . Data were normalized to GAPDH as a loading control. Primary antibodies used for detection were anti-P53 , anti-P21 , anti-BubR1 , anti-Securin , and anti-GAPDH . Secondary antibodies conjugated to HRP were used at 1∶2000 (Santa Cruz).After treatment with 1 µg/ml Nocodazole for 18 hours, total protein was extracted from approximately 5–10×10P<0.05 was considered significant.Chromosomes spreads were prepared from control, MDM4FL, MDM4S- expressing cells, and stained with Giemsa. Images were acquired with Motic high quality scientific grade CCD cameras (Hong Kong). Metaphase cells (75 per sample) from control, MDM4FL and MDM4S expressing pools were scored for chromosomes. Three independent chromosome counts were obtained for each data set, and the rank sum test used to compare chromosome number dispersion. Kruskal-Wallis was used to compare the medians of three ranked variables. All statistical analyses were performed using SPSS 16.0  and TP53, a male/female ratio of 1.14 (8∶7) and median age of 59 years , with seven patients (46.7%) ≥60 years. Two patients (13.3%) had WBC counts greater than 100×109/L. One patient was classified with M0, four with M2, five with M4 and five with M5 according to FAB classifications. Karyotype analysis showed monosomy 5 (−5) (n = 2), and monosomy 7 (−7) (n = 4). Of the 15 patients monitored for therapy response and survival, four achieved complete response (CR) and two achieved partial response (PR). The median survival time was 292 days . The clinical characteristics of the 15 CK-AML karyotypes were provided in P = 0.001) (This study cohort included 15 CK-AML patients with wild-type = 0.001) .TP53 mutation and deletions were detected by genome PCR sequencing and interphase FISH in 24 CK-AML cases, and 15 CK-AML cases were wild-type TP53. In order to rule out other genes mutation associated with poor prognosis of CK-AML, we detected RUNX1 mutations in 15 CK-AML patients and FLT3-ITD mutation in 131 de novo AML cases (15 patients with wild-type TP53 and 116 NK-AML patients.). Among the 15 CK-AML patients, no RUNX1 mutations were detected. The positive rates of FLT3-ITD in CK-AML and NK-AML were 20% and 23.2%, there was no significant difference between the two groups. (P>0.05).MDM4FL and MDM4S mRNA expression levels in CK-AML and NK-AML patients were assessed by real-time RT-PCR. The results indicated that normalized MDM4FL levels were 5.82 (1.67–20.28), while MDM4S levels were 22.25 (2.42–204.51). Both increased in CK-AML patients, with MDM4S showing a more notable increase (increase . The melincrease .P<0.05) (P<0.05) (P<0.05)  . We next(P<0.05) . Finally(P<0.05) .P<0.05), but it did not decline significantly in MDM4S-expressing cells (P>0.05). However, the p21 expression levels decreased in both MDM4FL and MDM4S-expressing cells compared with control (P<0.01)  .The spindle checkpoint proteins, BubR1 and Securin, were assessed by western blot in control, MDM4FL or MDM4S-expressing cells. The results showed that the expression levels of BubR1 and Securin in MDM4FL and MDM4S-expressing cells decreased following Nocodazole treatment. However, control cells exhibited increased Securin levels, consistent with previous reports P<0.05). Therefore, we conclude that at least one of these chromosome numbers had a different ranking distribution relative to the others. Boxplot analysis suggests that the MDM4S and MDM4FL cells most likely have different distributions from control cells. The chromosome numbers of each group reflects the range of chromosome numbers for MDM4S and MDM4FL, which were much more diverse. There were several singular and outlier values in MDM4FL or MDM4S-expressing cells, however they were not found in control cells . However, all myeloid cell lines either contain mutant p53 or do not express p53 BubR1 is a critical component of the spindle checkpoint. BubR1 performs several roles during mitosis and ensures accurate chromosome separation TP53. Although it is not well known if there is a causal relationship between MDM4 overexpression and aneuploidy, these date raise the possibility that MDM4 overexpression plays a role in CK-AML pathogenesis. It will be necessary to evaluate more patients and to further explore the molecular mechanisms of MDM4 overexpression and to develop targeted therapies for CK-AML patients. At least in theory, restoration of p53 function is a potential therapeutic approach in leukemia. Bista M et al Suppression BubR1 results in a dysfunction spindle checkpoint and leads to abnormal mitosis and aneuploidy MDM4 expression levels were elevated in CK-AML patients relative to NK-AML patients, MDM4-overexpressing HepG2 cell lines had a reduced mitotic index, reduced p21, BubR1 or Securin expression levels following Nocodazole treatment, and MDM4-overexpressing cells were aneuploidy or polyploidy. Based on data presented in this study, we speculate that the leukemogenic mechanism of CK-AML without TP53 alternations is partly due to the p53 signaling pathway inhibited and the spindle checkpoint weakened by MDM4 overexpression. MDM4 may be a novel therapeutic target in the treatment of CK-AML patients with wild-type TP53.


Using specimens from 41 patients with acute myeloid leukemia (AML), we studied the factors that might contribute to clinical response or resistance. For this purpose, thawed aliquots of primary AML samples were immunophenotypically characterized and subjected to various doses of AMG 330 in the presence or absence of healthy donor T-cells. After 48 hours, drug-specific cytotoxicity was quantified and correlated with CD33 expression levels, amounts of T-cells present, and other disease characteristics. AMG 330 caused modest cytotoxicity that was correlated with the amount of autologous T-cells (P = 0.0001) but not CD33 expression, as AMG 330 exerted marked cytotoxic effects in several specimens with minimal CD33 expression. With healthy donor T-cells added, AMG 330 cytotoxicity depended on the drug dose and effector:target (E:T) cell ratio. High cytotoxic activity was observed even with minimal CD33 expression, and AMG 330 cytotoxicity and CD33 expression correlated only at high E:T cell ratio and high AMG 330 doses (P<0.003). AMG 330 resulted in significantly higher cytotoxicity in specimens from patients with newly diagnosed AML than those with relapsed/refractory disease despite similar levels of CD33 on myeloblasts. AMG 330 cytotoxicity also appeared greater in specimens from patients with favorable-risk disease as compared to other specimens. Together, our data demonstrate that AMG 330 is highly active in primary AML specimens across the entire disease spectrum, while suggesting the presence of yet undefined, CD33-independent, relative resistance mechanisms in specific patient subsets.The CD33/CD3-bispecific T-cell engaging (BiTE) antibody construct, AMG 330, potently lyses CD33+ leukemic cells  Over the last several decades, the outcomes for many patients with acute myeloid leukemia (AML) have significantly improved. However, much of this progress is due to advances in supportive care, which have rendered curative-intent chemotherapy and allogeneic hematopoietic cell transplantation (HCT) safer and paved the way for intensified treatment algorithms –3. DespiAntibodies raised high expectations as a potent means of eliminating malignant cells with limited non-specific toxicities. Because of well-defined surface antigens and easy tumor cell accessibility, AML has been a paradigm for the therapeutic use of unconjugated and drug/toxin-loaded antibodies. Hitherto most exploited is CD33, a myeloid differentiation antigen displayed on at least a subset of AML blasts in most patients and, possibly, on leukemia stem cells in some , 5. Inde+ AML cell lines or primary human AML cells in the presence of healthy donor T-cells or autologous T-cells from AML patients at low effector-to-target (E:T) cell ratio , P = 0.0001). AMG 330 exerted marked cytotoxic activity in several specimens with very low CD33 expression levels on AML blasts, and we found no significant correlation between CD33 expression levels and drug-induced cytotoxicity  and the E:T cell ratio (e.g. P<0.0001 at 500 pg/mL). However, high activity of AMG 330 was even observed in specimens with very low CD33 expression on AML blasts. Similar to the experiments in which no healthy donor T-cells were added, there was no significant correlation between drug-induced cytotoxicity and CD33 expression levels on AML blasts at lower E:T cell ratios  than those with relapsed/refractory disease  as well as after 48 hours in cytokine-containing liquid culture (i.e. >30% viable cells). As these (arbitrarily defined) requirements could be criticized as not being stringent enough, we performed additional analyses in the subset of 25 primary AML specimens that showed a viability of >70% both at thaw as well as after 48 hours in cytokine-containing liquid culture . As summin vitro culture in the absence of AMG 330 treatment. As shown in in vitro culture relative to aliquots that were not treated with AMG 330.Finally, to test the effect of AMG 330 on immature AML cell populations, we incubated unsorted cells from 28 AML specimens with or without AMG 330 in the presence of healthy donor T-cells. After 48 hours, healthy donor T-cells were removed by FACS, and remaining patient-derived cells placed in liquid culture for 4 weeks. After 2 and 4 weeks, aliquots of cultured cells were then subjected to CFC assays, and CFU-GMs quantified after 10–14 days. A total of 16 specimens each yielded CFU-GM growth after 2 and 4 weeks, respectively, of + acute lymphoblastic leukemia (ALL) that persisted or relapsed after chemotherapy [Bispecific constructs that harness the immune system in the elimination of cancer cells are a long-pursued strategy to improve the efficacy of anti-tumor antibodies. Many bispecific construct modalities have been explored over the years, but their success was limited by suboptimal effector cell recruitment and challenges with large-scale, clinical-grade antibody production , 21. Undotherapy –25, sugg+ AML cells [With the expression of CD33 on myeloid blasts in most AML cases, and the survival improvement seen with GO in some patient subsets with this disease, AMG 330 is a logical first BiTE antibody construct for the treatment of human AML , 13. RecML cells –17; of nML cells . While tin vitro. As we were interested in discerning differences between AML cells from individual patients, we studied the effects of AMG 330 after a short incubation period  and at ll lines . Thus, oll lines , our curll lines , 27.4 molecules/AML blast [NPM1 as well as high allelic FLT3/ITD mutations, while expression is generally low with core-binding factor translocations [In myeloid cell lines engineered to overexpress various amounts of CD33, our previous studies demonstrated that AMG 330-induced cytotoxicity was proportional to the amount of CD33 expressed on AML cells . This obML blast , 30 and ML blast , 31. Comocations , 28, 32.The emerging clinical data with blinatumomab in ALL suggest that BiTE antibody constructs may be largely non-cross resistant to commonly used chemotherapeutics  as they can be effective in otherwise treatment-refractory patients –25. ConsAlthough our findings are significantly limited by the small number of specimens in this patient subset, we also observed higher activity of AMG 330 in the 5 specimens from patients with favorable cytogenetic or molecular disease features as compared to the other specimens in experiments in which healthy donor T-cells were added. Unfortunately, while cytogenetic profiling was complete, molecular profiling was incomplete in many of the studied specimens and some samples with favorable-risk features (e.g. NPM1 mutation without FLT3/ITD or CEBPA double mutation) may not have been correctly categorized, potentially limiting this analysis. Thus, although sample misclassification would likely lead to a reduction of the difference between favorable-risk and other specimens, further studies with larger sets of specimens may be required to confirm our findings.in vitro even in cases of very low CD33 expression on myeloid blasts. Lower activity of AMG 330 was observed in relapsed/refractory AML specimens (relative to newly diagnosed AML specimens) and perhaps intermediate- or adverse-risk disease specimens (relative to favorable-risk specimens) suggesting the potential presence of yet undefined, CD33-independent, relative resistance mechanisms in defined patient subsets. Further laboratory studies will be required to identify the nature of these resistance mechanisms. Nonetheless, our data suggest that AMG 330 is broadly active against human AML, and support our conclusion that it should be clinically explored in patients with AML and, by extrapolation, perhaps other CD33+ hematopoietic malignancies.In summary, our data show that AMG 330 causes potent dose- and T-cell-dependent cytolysis of primary human AML cells S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.