Small angle X-ray scattering (SAXS) and circular dichroism (CD) studies revealed that temperature in the range of 30–40 °C is capable of opening the G1 domain alone, as remaining domains are held together by the Ca2+-sensitive C-tail latch without any loss in the secondary structural content. Full opening of all domains of tail-less gelsolin, and retention of closed shape for G2–G6 gelsolin merely by heating, further substantiated our findings. The Ca2+/pH independent activity of gelsolin near physiological temperature brought out a query: whether gelsolin is always active, and if not, what might deactivate it? Earlier, PIP2 has been reported to render gelsolin inactive with no structural insight. Reduction in shape parameters and modeling revealed that PIP2 reverses the temperature induced extension of g1-g2 linker leading to a compact shape seen for Ca2+-free gelsolin. Similar results for partially activated gelsolin (by low pH or Ca2+ ions below 0.1 μM) imply that inside cells, depolymerization, capping, and nucleation of F-actin by gelsolin is regulated by the culmination of local Ca2+ ion concentration, pH, temperature and PIP2 levels.This is the first report describing temperature based initiation of gelsolin’s F-actin depolymerization activity, even in absence of free Ca Gelsolin is one of the main members of the gelsolin family of proteins capable of depolymerizing F-actin by achieving active dissociation of actin units in assembly and keeping the dissociated actin units capped. Additionally, gelsolin can retard growth rate of F-actin filaments by binding to the growing end. In a seemingly contrasting manner, gelsolin can bind two monomeric or G-actin molecules, and can induce polymerization or nucleation of F-actin. This makes gelsolin as an important regulator of actin assembly inside cells. The plasma form of gelsolin is the fourth most abundant protein in plasma where in presence of 1 mM free Ca2+, plasma gelsolin performs the function of rapidly depolymerizing F-actin released upon cell death or injury, and keeps it from re-polymerizing. Latter ability has been attributed to potential role of gelsolin in different biomedical problems5. Thus, decoding the shape-function relationship of this varied function multi-domain protein has remained exciting to structure biologists. Crystallographic studies, radiolytic foot-printing and SAXS data analysis based models support that in absence of Ca2+ ions, the six homologous domains of this protein are packed tightly in a compact shape, and availability of Ca2+ ions induce systematic opening of the domains away from each other to expose actin binding sites4. Taking a cue from an earlier work which concluded that low pH can override the need for Ca2+ ions, our SAXS experiments revealed that buffer pH~5 can induce the first i.e. G1 domain to open up from other five domains, just enough to allow the actin binding site between the G1 and G2 domain to be available3. Additionally, scattering shape reconstruction of the full length gelsolin and its C-tail truncated version in low pH and/or in presence of Ca2+ ions clearly established that the C-tail latch which held together the G2 and G6 domains was sensitive only to Ca2+ ions and low pH could not open or affect the latch. In the same study, it was shown that addition of substantially less amount of Ca2+ ions to pH 5 activated gelsolin could induce complete opening of this six-domain protein analogous to that seen by 1 mM free Ca2+ ions at pH 8, representing plasma conditions which re-confirmed the Ca2+-specificity of the C-tail latch of gelsolin3. The available biochemical and structural information summarizes that the compactly packed structure/shape represents inactive gelsolin, low pH can open G1 domain away from other domains which makes the protein partially active, and free Ca2+ ions can lead to a completely open structure/shape which can perform all stages of actin assembly regulation.Shape-function studies on the six domain actin-assembly regulating protein, gelsolin has brought forth interesting findings, particularly how the compactly packed domains open up completely upon Ca°C, safely presuming that primary shape-function properties of gelsolin are regulated by only Ca2+ ions or low pH. Additionally, most of the crystal structures of gelsolin and its truncated form(s) +/− actin have been grown either at low pH or in temperature 4–24 °C (PDB ID/Temperature (°C): 1D0N/18, 1P8X/4, 1NPH/20, 1P8Z/20, 1RGI/4, 2FF3/20, 2FH1/4, 3FFK/24, 3FFN/24). Even our SAXS data collection of gelsolin −/+ Ca2+ and/or low pH were done at 10 °C5. In literature, we found only one study which experimentally compared the effect of temperature on functionality of gelsolin, i.e. how much Ca2+ ions are required by gelsolin to exhibit F-actin depolymerization activity6. These authors reported that at 37 °C, Ca2+-gelsolin depolymerization of pyrene labelled F-actin was detectable at 0.5 µM Ca2+ which reached half maximal at 2.2 µM Ca2+ and kept increasing till 0.1 mM Ca2+. In comparison, authors reported that at 24 °C depolymerization activity of gelsolin could be observed only at 10 µM Ca2+ which reached half-maximal 19 µM Ca2+ suggesting need for higher Ca2+ levels at lower temperature. Importantly, their results observed at 24 °C correlated well with previous reports done under similar conditions. This ten-fold difference in Ca2+ requirement at 24 vs. 37 °C clearly indicated that there is a role of increase in temperature in stabilizing the F-actin binding and depolymerizing competent conformations of gelsolin which overrides/complements the Ca2+-induced effects, though the authors mainly connected their findings to the role of Ca2+-sensitive C-tail region of gelsolin in enabling functional shape of full-length gelsolin and its variants.Interestingly, almost all the biophysical studies leading to above conclusions were done at temperatures below 37 7. However, this increase in activity was again attributed to Ca2+ ion sensitive C-tail latch, as the dynamics of tail-less gelsolin matched with adseverin . In current work, while experimentally evaluating influence of temperature on F-actin depolymerization of gelsolin, we found that this protein can depolymerize F-actin when heated to temperatures close to 35 °C even in absence of Ca2+ ions or low pH albeit lesser than Ca2+-induced effects but comparable to influence of low pH. Experiments were repeated to confirm the observations followed by shape analysis using SAXS experiments which brought forward that the essential and minimal step to be depolymerization competent i.e. opening of G1 domain of gelsolin can be achieved by increase in temperature.Interestingly, a comparative study done for gelsolin and adseverin reported that with an increase of mere 10 °C in temperature the actin depolymerisation activity of gelsolin increased by 8 folds2+ ions or low pH can enable gelsolin to depolymerize labelled F-actin which can be recorded as a decrement in pyrene fluorescence8. Yet most of the previous experiments, in presence or absence of Ca2+ ions and pH close to 7.5 or at low pH in absence of Ca2+ ions were done at temperatures close to or below 25 °C, except one study where the depolymerizing activity of Ca2+-gelsolin was also compared at 37 °C6. To test dependence of F-actin depolymerization activity of gelsolin on temperature, heated samples of gelsolin, in buffers having 1 mM free Ca2+ (pH 8)  and pH 5 (middle) and pH 8 (bottom), were added to pyrene-labelled F-actin. In the control assays, only equivalent amounts of buffer were added, and all datasets were normalized to these read-outs from F-actin alone. Results showed that Ca2+-gelsolin heated up to 40 °C retained F-actin depolymerizing activity, but exposure to higher temperatures gradually reduced efficacy of Ca2+-gelsolin with almost complete loss by 60 °C . To the best of our knowledge, these experiments provided first direct indications that temperature alone can somehow allow gelsolin to achieve a shape which can depolymerize F-actin.To our sheer surprise, increasing the temperature of gelsolin in absence of Ca°C before associating into higher order aggregates as seen in the SAXS profiles collected at 60, 70 and 80 °C  is directly proportional to square of the mass of the scattering species, and knowing that protein concentration remained comparable (at least before onset of higher order aggregation), we used I0 values estimated from Guinier approximations to estimate mass of the predominant scattering species in solution at different temperatures  values calculated from the slope of the linear region of the Guinier approximation for the samples at different temperatures have been plotted as a function of the temperature  calculations were done to assess the conformations accessible to proteins in solution Fig. . Against2+ ions, no free Ca2+ ions but pH of 5 and no free Ca2+ ions and pH of 8 to see change in ellipticity values as a function of temperature. In all three conditions, as in SAXS experiments, protein aggregation was visible from 60 °C onwards, and it correlated with earlier reports10. Considering the data prior to initiation of aggregation i.e. below 60 °C, the slow decrement in secondary structural content had a mid-point close to 45 °C for all three conditions  curves from samples (8 mg/ml) at 10, 30 and 40 °C are shown in Fig. °C in buffer having pH 8 and 1 mM EGTA or under Ca2+-free conditions. The dummy atom model computed for 10 and 20 °C compared well with the crystal structure of Ca2+-free gelsolin (PDB ID: 3FFN one chain) (model of 20 °C is not shown)  value for the ten models are mentioned next to each SAXS data based model. NSD values close to 1 support that all individual models resemble each other and can be reliably averaged.Pairwise distance distribution function (PDDF), P(r) is the frequency distribution of interatomic vectors which best-represents the scattering profile emerging from the molecules in solution. In this work, all the PDDF calculations were done in automated manner considering monodisperse globular nature of the scattering species. Probability of finding vectors corresponding to 0 nm and a dimension equal to the longest vector in the scattering shape were considered to be zero. As described above, Iwn) Fig. . The ave°C showed that the gelsolin molecule appears to open from one side  clearly fitted in the extended out volume of the SAXS data based model  the extended away G1 domain and g1–g2 linker exposes the actin binding epitopes. These results further concluded that C-tail latch of gelsolin is specifically sensitive to Ca2+ ions, and explained the surprising F-actin depolymerizing activity of gelsolin in absence of Ca2+ ions upon heating.The model solved for gelsolin at 30 ide Fig.  which op °C Fig. . Being adel Fig. . Differe2+ ions sensitive C-tail latch, we studied temperature dependent SAXS profiles of two additional constructs of gelsolin: (1) ΔCT gelsolin – gelsolin lacking the C-terminal latch (sequence terminated at S728 followed by six Histidine tag), and (2) G2–G6 gelsolin – gelsolin lacking G1 domain (the construct started from L162 preceded by a methionine). Variable temperature SAXS data was acquired on both the proteins at concentrations close to 8 mg/ml, and variations in the RG values were plotted as a function of temperature  in buffer lacking free Ca2+ ions and having pH 8 was heated to different temperatures and then the temperature was brought back to 10 °C, while acquiring SAXS data at different temperatures. Guinier analysis provided the RG values of scattering particles which indicated that gelsolin molecules can reversibly close if they are heated up to 40 °C  did not show decrement in shape parameters upon cooling indicating irreparable or irreversible changes are initiated in the gelsolin proteins at these temperatures. Earlier, we had seen an increase in apparent molecular masses of the molecules at these temperatures which indicated some extent of association. In summary, our data indicated that gelsolin molecules open-up at temperatures close to physiological ones which somewhat correlates with enhanced activity reported earlier6.Quick query was raised if the temperature induced opening of gelsolin is a reversible phenomenon since previous characterizations of gelsolin under Ca °C Fig. . SamplesIf the above observations are true in biology then gelsolin may never be inactive as even in absence of Ca2+ions or low pH, gelsolin can achieve an actin binding shape, and thus interfere in actin assembly process. Situation can get further complicated since there would be a very likely interplay of temperature and available Ca2+ ions or low pH in surroundings of gelsolin molecules. As published before by us, in low pH buffer significantly less amount of Ca2+ ions were required to open up the full gelsolin molecule as the C-tail latch is sensitive to Ca2+ ions only3. In this work, we showed that C-tail latch remains unaffected by temperature, and cutting off C-tail latch allows temperature alone to open-up all domains of gelsolin. We propose that physiological temperature in conjunction with the low levels of Ca2+ ions (below 0.1 μM2) would be able to completely open gelsolin molecules for their actin assembly regulation function. Outside cells, in plasma, large concentration of free Ca2+ ions (~1 mM) would invariably activate gelsolin into a completely open shape11. In other words, considering the fact that gelsolin can be activated by Ca2+ ions, and/or low pH, and/or (now) physiological temperature, it implies that gelsolin would always be constitutively active both inside cells and outside, unless there is a molecule which deactivates it, fully or partially.2) was shown to deactivate gelsolin molecules though no direct structural evidence is available to date13. Gel filtration and intrinsic tryptophan fluorescence experiments provided evidence that gelsolin binds PIP2 and their affinity is increased by the presence of µM levels of free Ca2+ ions and the need for Ca2+ ions is reduced upon lowering of buffer pH14. Biochemical data have shown that PIP2 predominantly binds to the N-terminal half of gelsolin, a portion which is highly conserved amongst the gelsolin superfamily, and to date only one structure (NMR based) is available which shows how the peptide corresponding to residues 150–169 of gelsolin binds to PIP2 (PDB ID: 1SOL)18. The mechanism of action of PIP2 and gelsolin inactivation remains debatable, as one notion is that the PIP2 competes with gelsolin bound actin for binding to g1–g2 linker of gelsolin, which gets challenged since PIP2 binding epitopes are somewhat occluded in bound actin state19. Based on biochemical studies, another mechanism has been composed which suggests that PIP2 binding dislodges Ca2+, leading to release of bound actin to cytoplasm followed by closure of the g1–g2 linker which brings G1 and G2 in proximity of each other20. Furthermore, the cells with increased PIP2 have an altered non-native phenotype similar to that of gelsolin null phenotype which reflects uncontrolled association state of actin inside cells21. In summary, PIP2 is known to bind the residues in the end of the G1 domain and the g1–g2 linker which become available only when the g1–g2 linker opens up from the compact shape of the inactive gelsolin. Since temperature induced activation of gelsolin also involves extension of g1–g2 linker, the known site for PIP2 binding, we decided to explore: (1) if PIP2 can deactivate temperature-activated gelsolin and (2) whether these events can be tracked by SAXS data analysis which may explain vital role of PIP2 in regulating “apparently always” active gelsolin at least inside cells.In literature, phosphatidylinositol 4, 5-bisphosphate  and G2–G6 domains from crystal structure of Ca2+-free gelsolin , could be fitted reasonably well  up to a pCa value of 7.1 or 0.1 µM. For higher concentrations, a decrease for visible but it was insignificant after free Ca2+ concentrations surpassed 10 µM  can induce partial opening of gelsolin where G1 domain moves away from C-tail locked G2–G6 domains. We showed in this work that in absence of these two factors, increase in temperature to 35–40 °C can also open and stabilize G1 domain away from the other five locked domains analogous to that induced by low pH. Based on our previous work and complementary work from other groups, there was a query that in physiology, in absence of Ca2+ ions or low pH, gelsolin adopts an inactive resting shape? Our present shape information in conjunction with results reported earlier indicate that very likely, at physiological temperature, intracellular gelsolin exists in this partially open form and the inactive compact shape may never exist except in availability of PIP2 which binds to g1–g2 linker and induces shape changes which bring G1 domain close to G2 domain. In contrast, the high concentrations of free Ca2+ ions dictate the predominant shape of the gelsolin molecule. These scenarios are possible in plasma where free Ca2+ concentration is close to 1 mM and under injury or stress conditions leading to rise in intracellular Ca2+ concentration. Essentially, the high Ca2+ ion concentration  will lead to the fully active state of gelsolin due to additional factors of low pH or temperature which will enable rapid remodelling of F-actin assembly where the latter is essential for many cellular functions. Another scenario could be the release of Ca2+ ions from the endoplasmic reticulum (ER) membranes, which is released only when a signal is received in the form of inositol triphosphate (IP3) molecule. Interestingly, IP3 molecule is the product of PIP2 hydrolysis by Phospholipase Cγ1 (PLCγ1)24 which suggests a correlation between local PIP2 and Ca2+ levels. Gelsolin and its activation by low pH was discussed by us in view of lowering of pH during apoptosis3. We found one publication which linked apoptosis and IP3-linked mitrochondrial Ca2+ signals25 which indicates that PIP2 and Ca2+ and pH could be interconnected in regulating gelsolin’s function.Shape-function studies in this work brought forth a new physiological activator of gelsolin lue Fig. . Trace a2 is primarily located at the cell membrane as well as perinuclear spaces, however the PIP kinase mediated synthesis of PIP2 in plasma membrane is also reported27. It is also proposed that certain proteins might serve as “buffers” which bind and sequester PIP2 in the plasma, and subsequently concentrate PIP2 in lateral membrane domains26. Gelsolin has been suggested to weakly bind PIP2 which suggests that PIP2 may get released at some point. As of today, it is clear that inactive gelsolin cannot bind PIP2, but there is no data which supports a differential affinity of PIP2 with open vs. closed shape of gelsolin. Thus, the precise mechanism of release of PIP2 remains unclear. For now, we opted to use SAXS data analysis and modeling to interpret the unexpected F-actin depolymerization results as a function of temperature as this technique is not limited by the need for a diffraction quality crystal or extensive timelines for labelling and/or imaging based methods. Moreover, SAXS experiments can aid in tracking shape changes in protein molecules albeit experiments are planned properly and results are supported by orthogonal approaches as we did by developing and studying mutant proteins. Considering the new queries raised and their implications in biology, we intend (and invite other researchers) to extend these findings in cell line based experiments and understand how the shape changes affect the signalling pathways associated/dependent on gelsolin. Thus, temperature plays a key player inside cells in activating shape of gelsolin which in turn regulates multiple functions related to assembly state of actin, and flux of available PIP2 (which in turn is regulated by Phospholipase C) controls active state of gelsolin by specifically closing the g1–g2 linker.Interestingly, PIPhttp://web.expasy.org/protparam/).Different gelsolins used in this work: full-length gelsolin, gelsolin lacking C-tail latch (ΔCT), and gelsolin lacking G1 domain (G2–G6) were expressed in vectors and cells as published before refs 8. Briefly, pyrene labelled G–actin was converted to F–actin by adding 3 mM MgCl2 and 100 mM KCl in presence of gelsolin (in Actin:Gelsolin molar ratio of 500:1) followed by overnight incubation at 20 °C. To measure depolymerization capability, stock of pyrene-F–actin (9.88 µM) was diluted to 100 nM in F–buffer . For activity assays under Ca2+-free conditions, 0.2 mM CaCl2 in F–buffer was replaced with 0.2 mM EGTA. To understand the effect of temperature on the activity of gelsolin, vials containing 50 μl of solution of gelsolin (1.4 mg/ml or 17 μM) was heated at the desired temperature (in the range of 10 to 80 °C) for 30 minutes MyGeneTM Series Peltier Thermal cycler . Subsequently, the protein solution was diluted to 1.8 μg/ml (20 nM) in buffers of respective pH, kept at 25 °C and used for depolymerization assays which were done at 25 °C. It is worth noting here that same gelsolin concentration was used for observing/comparing depolymerization ability of gelsolin as a function of Ca2+ ions or pH in buffer. The fluorescence assays were performed in a 96 well flat bottom FlouroNunc plates. In each well, total reaction volume was close to 200 µL which included gelsolin and F–actin in a molar ratio of 1:10 (Gelsolin: F–actin). Importantly, F–actin was added last in the wells and readings were taken within 15–20 seconds of final addition. For measurements, excitation wavelength was kept at 365 nm and emission was recorded at 407 nm using Tecan plate reader with i-control software . The numbers of flashes were kept at 25 with an integration time of 20 µs. Each sample was assayed in duplicates, and the average of reading of both the samples was taken as final readout.The rate of decrement in fluorescence of pyrene–labelled F–actin in presence of gelsolin was used to determine the ability of gelsolin to depolymerize actin filaments as done before°C, SAXS data was collected on gelsolin solution (about 30 μl) and matched buffer from 10 to 80 °C. Line collimation was used for incident X-rays on sample filled in a quartz capillary of 1 mm diameter for 30 minutes. Scattered X-rays were recorded on a 1D CMOS Mythen detector (Dectris Switzerland). SAXS datasets for sample and buffer were processed using SAXStreat and SAXSquant softwares. Using the beam profile, the data collected on line collimation was desmeared to represent scattering arising from point collimation. Intensity of scattering, I(Q) was obtained as a function of Q, where Q was considered as 4 π sin θ/λ. All processing conditions were identical for all datasets, and I(Q) profile of the respective buffer was subtracted from the I(Q) profile of the solution to obtain SAXS profile arising from the protein molecules in solution. SAXS data profile(s) of gelsolin at different temperatures were analyzed using ATSAS 2.7.1 pipeline of programs to obtain information about scattering size and shape of the protein molecules at that temperature. Briefly, Kratky analysis, Guinier approximations and automated estimations from probability distribution of interatomic vectors were done using the suite of programs.To study the effect of varying temperature on the global shape of gelsolin, SAXS datasets were collected using SAXSpace instrument . At temperature intervals of 5 or 10 G values of the various conformations accessible to the gelsolin molecules at different temperature. This genetic algorithm based program presumes the observed shape profile from the scattering species in solution to be a weighted average of ensemble of conformations accessible to the molecule in solution, and attempts to describe the experimentally acquired SAXS profile as an ensemble of models with varying RG values. Earlier, it has been successfully applied for flexible proteins to map the RG values of the conformations accessible to proteins in solution28. We have applied this protocol to address ensemble of conformations accessible to a well-known inherently disordered protein, Calmodulin29. For this work, against the random pool of structures, optimized ensemble of 5000 conformers were generated by performing 100 independent runs of 50 conformers each. Using the SAXS profiles of the full-length gelsolin at different temperatures, and SAXS data of ΔCT gelsolin and G2–G6 gelsolin at different temperatures, EOM calculations were done to estimate RG values of the conformations in the ensemble.Using the experimental SAXS data as reference and the EOM program in the ATSAS 2.7.1 suite, calculations were done to estimate the R°C, with a temperature ramp of 3 °C/min. From the complete scans, ellipticity values recorded at 222 and 218 nm were plotted to compute melting temperature, Tm or the temperature at which half of the α-helical or β-sheet content was unfolded during the acquisition time of the data.To gain insight into the secondary structural content in gelsolin molecules and their change as a function of temperature, CD experiments were carried out using Jasco spectrophotometer 815 (Tokyo Japan). The temperature for the experiments was controlled with a Peltier type temperature control system, model PTC-424S. A quartz cell with 0.2 cm path length was used and spectra were recorded from 205 to 250 nm at intervals of 1 nm. For each sample and buffer, 5 scans were averaged and final spectral profile of gelsolin was obtained by subtracting profile of the buffer. Temperature scans were done with temperatures starting from 25 to 90 30, albeit with slight modifications. Briefly, a 10% non-denaturing gel was cast (pH 8.8), and this gel was pre-run at 10 V/cm for one hour at 4 °C before loading samples. After pre-run, the electrode buffer was discarded and replaced with fresh buffer. Samples of FPLC purified gelsolin at concentration same as that used for SAXS experiments (8 mg/ml) were individually heated from 10 to 80 °C for 30 minutes and cooled back to 25 °C in a thermal cycler. From these vials 2 μL (16 μg per lane) of protein mixed with 2x non-denaturing gel loading dye (pH 8.8) and loaded in the wells of on the native gel. Post-run, the gel was stained with 0.25% Coomasie Brilliant Blue R – 250, followed by de-staining and image was captured using AlphaImager .The continuous native PAGE was performed as described earlier31. Dummy aspartic amino acid residues equal to mass of the protein molecules were considered to model the shape of gelsolin and its variants. Ten models were generated for each dataset, and they were averaged using DAMAVER suite of programs. Essentially, for each set, the models were superimposed over a reference model by aligning inertial axes using SUPCOMB20 program from ATSAS suite, and their NSD values were calculated. Any models with NSD value more than twice the standard deviation is rejected by the averaging program. In this work, none of the models were rejected being very similar to each other in the sets, except one of the ten for ΔCT gelsolin at 35 °C. The averaged models are presented in the Figures, and were compared with crystal and modelled structures. Open source version of Pymol was used for visual analysis of results and figure generation presented in this publication32.Scattering shapes of gelsolin or versions at different temperature point were reconstructed using chain-like ensemble modeling program GASBOR in reciprocal space using SAXS I(Q) profile and results from its pair wise distribution of interatomic vectors2 was prepared . This mixture was used to acquire SAXS data collection in the temperature range of 10–45 °C to monitor if any shape changes occur to gelsolin pre-mixed with PIP2. Additionally, PIP2 was mixed with gelsolin sample (~3 mg/ml) heated to 40 and 50 °C, respectively, and the sample was cooled back while measuring SAXS data at different temperatures. The SAXS data from these samples were analyzed as described above.For this experiment, 1:1 molar mixture of gelsolin with PIPSupplementary Information


Transient receptor potential (TRP) ion channels have emerged as cellular sensors for thermal, chemical and mechanical stimuli and are major contributors to Ca2+ signaling, playing an important role in diverse physiological and pathological processes. Notably, TRP ion channels are also one of the major downstream targets of Ca2+ signaling initiated either from TRP channels themselves or from various other sources, such as G-protein coupled receptors, giving rise to feedback regulation. TRP channels therefore function like integrators of Ca2+ signaling. A growing body of research has demonstrated different modes of Ca2+-dependent regulation of TRP ion channels and the underlying mechanisms. However, the precise actions of Ca2+ in the modulation of TRP ion channels remain elusive. Advances in Ca2+ regulation of TRP channels are critical to our understanding of the diversified functions of TRP channels and complex Ca2+ signaling.Ca Transient receptor potential (TRP) ion channels are a large family of cation channels consisting of six mammalian subfamilies: TRPV, TRPM, TRPA, TRPC, TRPP and TRPML . They ar2+ absorption and homeostasis i) and subsequent Ca2+ signaling. Ca2+ is a ubiquitous second messenger participating in numerous signal transduction pathways involved in biological processes as diverse as secretion, gene transcription and cell death i due to channel opening, loads Ca2+ onto N-lobe. Ca2+-loaded N-lobe then switches to bind to a second C-tail from another subunit leading to channel inactivation. Indeed, a TRPV6 mutant (L707A) enhanced N-lobe binding and also promoted TRPV6 inactivation i by Gq-PLC-coupled receptors also activate TRPA1. TRPA1 may also be potentiated by Ca2+ influx through other nearby Ca2+-permeable channels, such as TRPV1, that form a complex with TRPA1 i is both necessary and sufficient for activation of both channels [2+]i i ,111.2+]i between 0.3 μM and 1 μM, similar to TRPM4, but inhibited at higher [Ca2+]i (≥1 μM) [2+ levels [2+]i was rapidly followed by pronounced channel inactivation or desensitization, leading to the distinctive transient activation of the channel [2 and was suggested to be Ca2+-dependent [2+-independent process [2, a positive regulator of TRPM4 [2+ entry through other TRP channels  has been shown to be sufficient to induce PIP2 hydrolysis, it is therefore likely that PIP2 hydrolysis, as a consequence of Ca2+-triggered activation of PLC, contributes to the desensitization of TRPM4 and TRPM5.TRPM5 was shown to be activated by [Ca (≥1 μM) , which i+ levels . Further channel , which i channel . The rapependent ,112, tho process . TRPM4 cof TRPM4 ,113. As 2+ sensitivity of TRPM4. The first mechanism involves CaM, similar to other TRP channels. This is evidenced by the finding that Ca2+ activation of TRPM4 is markedly decreased by co-expressing the dominant negative CaM1234 and that direct application of CaM to excised TRPM4 channels prevents TRPM4 desensitization [2+ activation of TRPM4 [2+ to two negatively charged residues (D1049 and E1062) near and in the TRP domain. Neutralization of these residues reduced Ca2+ sensitivity [2+ binding site is within the intracellular side of the S1–S4 domain [2+ sensitivity is the direct action of Ca2+ on TRPM5, independently of CaM, because Ca2+ is sufficient to induce TRPM5 activity in excised patches and CaM inhibitors have been shown to have no effect on the Ca2+ activation of TRPM5 [2+ binding sites on TRPM5 have not yet been identified.Two other different mechanisms are also involved in the Catization . In an aof TRPM4 . The secsitivity . However4 domain . For TRPof TRPM5 ,110. HowTRPM8 channels are activated by cold temperatures (≤30 °C) and cooling compounds, such as menthol, acting as a cold sensor  and sett2+, and Ca2+ influx upon channel opening triggers rapid channel desensitization [2+, TRPM8 currents are substantially larger and lack desensitization [2+ acts by mainly activating Ca2+-sensitive PLCδ4, leading to hydrolysis of PIP2, a regulator essential for maintaining channel activity [2+-dependent desensitization of TRPM8. Nevertheless, PLCδ4-lacking mice exhibited increased duration and events of TRPM8-dependent nocifensive behaviors in response to cooling [2 underlies TRPM8 desensitization and cold adaptation in vivo.TRPM8 is permeable to Catization , a procetization . It was activity ,122,123. cooling . These e2+ [2+ is therefore important for elucidating the mechanisms of ADPKD.TRPP2 is also known as polycystin-2 (PC2) or PKD2 and is permeable to Ca2+ . Mutatio2+ . Underst2+ exerts a dual effect on TRPP2 with lower concentrations of Ca2+ (≤0.3 μM) facilitating, and higher concentrations of Ca2+ inhibiting, the channel, leading to a bell-shaped Ca2+ dependence [2+ sensitivity of TRPP2 channels is believed to be due to a direct binding of Ca2+ to the channel. Indeed, a single Ca2+ binding site was revealed by NMR in the EF-hand domain (720–797) in the C terminus of TRPP2 [2+ binding and rendered the mutated channel inactive [2+ sensor. On the other hand, phosphorylation of TRPP2 at S812 close to the EF-hand domain by casein kinase II (CK2) increased the Ca2+ sensitivity of TRPP channels, because mutation of S812 markedly reduced Ca2+-dependent activation and inactivation of TRPP2, leading to a positive shift in Ca2+-dependence [2+ to the EF hand domain. However, the single Ca2+ binding site in the EF hand domain cannot account for the dual effects of Ca2+ on TRPP2. Therefore, Ca2+-dependent regulation of TRPP2 likely involves other unknown mechanisms.Capendence . The Ca2of TRPP2 . Mutatioinactive ,130, sugpendence . Presuma2+ signaling is necessary for many physiological processes. TRP ion channels are not only the major contributors to Ca2+ signaling, but also are the targets of Ca2+ signaling, ensuring rapid control of functions of TRP ion channels and associated Ca2+ signaling. Ca2+-regulation of TRP ion channels largely involves three major mechanisms: direct binding of Ca2+ to channels, Ca2+-sensing CaM and Ca2+-dependent hydrolysis of PIP2. Of them, CaM is probably the most common mechanism in mediating Ca2+-dependent regulation of TRP ion channels. CaM not only directly senses different levels of Ca2+ through two different lobes, but also serves as a multifunctional effector protein, providing vast versatility for decoding Ca2+ signals and modulating TRP ion channels. Future research is required to further understand diversified regulation of TRP ion channels by CaM. Accumulating evidence also suggests the presence of other Ca2+-dependent mechanisms critical in the regulation of TRP ion channels, which remain to be investigated.Spatial and temporal control of Ca


We tested whether loss of physiological Ca2+ transients is an initial cause leading to mitochondrial dysfunction in denervated skeletal muscle.Motor neurons control muscle contraction by initiating action potentials in muscle. Denervation of muscle from motor neurons leads to muscle atrophy, which is linked to mitochondrial dysfunction. It is known that denervation promotes mitochondrial reactive oxygen species (ROS) production in muscle, whereas the initial cause of mitochondrial ROS production in denervated muscle remains elusive. Since denervation isolates muscle from motor neurons and deprives it from any electric stimulation, no action potentials are initiated, and therefore, no physiological CaA transgenic mouse model expressing a mitochondrial targeted biosensor (mt-cpYFP) allowed a real-time measurement of the ROS-related mitochondrial metabolic function following denervation, termed “mitoflash.” Using live cell imaging, electrophysiological, pharmacological, and biochemical studies, we examined a potential molecular mechanism that initiates ROS-related mitochondrial dysfunction following denervation.2+ level inside mitochondria follows the time course of the cytosolic Ca2+ transient and that inhibition of mitochondrial Ca2+ uptake by Ru360 blocks the effect of electric stimulation on mitoflash activity.We found that muscle fibers showed a fourfold increase in mitoflash activity 24 h after denervation. The denervation-induced mitoflash activity was likely associated with an increased activity of mitochondrial permeability transition pore (mPTP), as the mitoflash activity was attenuated by application of cyclosporine A. Electrical stimulation rapidly reduced mitoflash activity in both sham and denervated muscle fibers. We further demonstrated that the Ca2+ transients due to denervation results in the downstream absence of mitochondrial Ca2+ uptake. Our studies suggest that this could be an initial trigger for enhanced mPTP-related mitochondrial ROS generation in skeletal muscle.The loss of cytosolic CaThe online version of this article (doi:10.1186/s13395-017-0123-0) contains supplementary material, which is available to authorized users. Skeletal muscle is responsible for voluntary movements of the entire body. Because it comprises around 40% of whole-body lean mass of a human, skeletal muscle is also essential for maintaining the homeostasis of the whole-body metabolism . Skeleta2+ as a messenger to control events ranging from activation of contraction to cell death. Defective intracellular Ca2+ signaling has been linked to skeletal muscle dysfunction during aging  inside mitochondria upon voltage-induced Ca2+ release  . The change in fluorescence intensity F/F0 (t) of x-rhod-1   provided a recording of mitochondrial Ca2+ uptake represented by the ratio of f2/f1 . Both quantified cytosolic and mitochondrial Ca2+ transients induced by the elongated depolarization pulse are depicted in Fig. 2+] and the black trace for mitochondrial [Ca2+]). During the elongated depolarization, there was a delay for mitochondrial Ca2+ transient to reach its peak value. However, the mitochondrial Ca2+ transient was synchronized with the cytosolic Ca2+ transient. The data demonstrates that the Ca2+ level in mitochondria indeed follows the dynamic changes of the Ca2+ level in cytosol. In the case of denervation, no action potential is initiated by the motor neurons, thus there are no cytosolic Ca2+ transients, and therefore, no mitochondrial Ca2+ transients either. It is possible that dynamic alterations of mitochondrial Ca2+ transients may be essential to maintain the normal mitochondrial function in skeletal muscle. The absence of dynamic alterations of mitochondrial Ca2+ transients may be associated with the enhanced mitoflash activity, which is linked to the enhanced mitochondrial ROS generation following the denervation.We next examined whether mitochondrial Can = 5, 11, p < 0.001). In the second set of experiments, we tested whether the electric field stimulation could reduce the fluorescent intensity of MitoSOX Red in the denervated FDB muscle fibers. The denervated FDB muscle fibers were first set to a glass bottom culture dish. A few denervated muscle fibers were selected to receive the electric field stimulation. Immediately after the electric field stimulation, the MitoSOX Red was added into the dish following the same dye loading procedure. The MitoSOX Red intensity of the denervated muscle fibers was then evaluated. As shown in Figs. n = 9, p < 0.001 compared to the fibers without field stimulation). This set of data suggest that mitochondria in denervated skeletal muscle indeed response to the physiological intracellular Ca2+ transients with reduced superoxide production.Because of the ongoing debate of the mt-cpYFP sensitivity on ROS and pH , we used2+ level in mitochondria followed the dynamic changes of cytosolic Ca2+ level and Ru360 blocked the effect of electric stimulation on mitoflash activity , the denervated FDB fibers were loaded with MitoSOX Red for evaluation of mitochondrial superoxide level. As shown in Fig. n = 5, p < 0.05 compared to the denervated fibers without Ru360), suggesting that the transient mitochondrial Ca2+ uptake is likely responsible for suppressing the mitochondrial ROS production during a physiological Ca2+ release transient. We also conducted a control experiment to test if Ru360 has a potential effect on mitochondrial ROS production. As demonstrated in Fig. Since our voltage-clamp data demonstrated that Ca2+ signaling regulate mitochondrial ROS production in skeletal muscle mainly focused on the effect of a steady state level of Ca2+ inside mitochondria [2+ by mitochondria may also be a key player in maintaining the physiological function of mitochondria. We showed that the time course of free Ca2+ level inside mitochondria follows the time course of cytosolic Ca2+ transient and that physiological changes in mitochondrial Ca2+ levels could be essential for maintaining the functional integrity of mitochondria. Following denervation, there is no action potential initiated in muscle fibers, and therefore, no Ca2+ transients in the cytosol and mitochondria. Mitochondria respond to this condition with increased ROS production that is associated with mPTP opening. Restoring the physiological Ca2+ transients by electric stimulation reduced the mitochondrial ROS production and stopped the repetitive mPTP opening in the denervated muscle fibers.Previously published studies on how Cachondria . Here, w2+ level in the cytosol and mitochondria are implicated in this process. Here, we provide evidence that physiological Ca2+ transient is required to maintain the integrity of mitochondrial function in the physiological condition. Absence of the physiological Ca2+ transients is likely a direct cause of enhanced mitochondrial ROS production that is associated with the repetitive opening of the mitochondrial mPTP.Motor neuron innervation is critical for the growth and maintenance of muscle fibers. Denervation is known to cause muscle atrophy, manifested in age-dependent sarcopenia and other neurological diseases, such as amyotrophic lateral sclerosis (ALS). Studies by other investigators have shown that denervation leads to compromised EC coupling machinery in skeletal muscle  and alteWang et al. developed a mitochondrial-targeted, circularly-permutated yellow fluorescent protein (mt-cpYFP) as an indicator of ROS production and energy metabolism of individual mitochondrion in live cells including skeletal muscle cells –27. TheiIn both skeletal and cardiac muscle cells, mitochondria form a structural and functional network , 45, 46.2+ during skeletal muscle contraction [2+ uptake seems to be a double-edged sword for the fate of cells [2+ uptake is a physiological stimulus for ATP synthesis [2+ level leads to a coordinated upregulation of oxidative phosphorylation machinery, resulting in higher mitochondrial ATP output to meet the energy demanding of the cells [−/−) with global knockout of mitochondrial calcium uniporter (MCU) showed a smaller body size and impaired skeletal muscle performance along with absence of mitochondrial Ca2+ uptake in isolated skeletal muscle mitochondria, indicating that mitochondrial Ca2+ uptake plays an important role in skeletal muscle development and performance [2+ overload in mitochondria is also a pathological stimulus of ROS generation [2+ level, that in turn overloads mitochondria to stimulate the ROS production [2+ signaling regulate mitochondrial ROS production in skeletal muscle mainly focused on the effect of a steady state level of Ca2+ inside mitochondria, it is not known whether the dynamic Ca2+ uptake by mitochondria has a role in maintaining the mitochondrial functional integrity. Recording of the mitoflash signal in a skeletal muscle fiber following a short period of denervation allowed us to discover the early response of mitochondria to the absence of physiological cytosolic Ca2+ transients, which limits mitochondrial Ca2+ uptake and leads to an excessive increase in mitochondrial ROS production. Our result is in line with the study by Csordas et al. [3R-mediated Ca2+ transfer to mitochondria that stimulates oxidative metabolism in a cultured mammalian cell line [2+ transients in denervated muscle fibers and that the transient mitochondrial Ca2+ uptake could be a key regulator of mitochondrial mPTP activity. Interestingly, recent studies from other research groups also provided evidence of MCU-dependent mitochondrial Ca2+ uptake in protecting denervation-induced skeletal muscle atrophy by using virus-mediated overexpression of MCU [2+ overload into mitochondria leads to mPTP opening and ROS production, one of the major findings in the present study is that the denervation-induced ROS elevation could be reduced by electrical stimulation, indicating an unexpected role of the Ca2+ uptake into mitochondria for control of ROS production. While a consistently elevated intracellular Ca2+ level leads to the Ca2+ overload in mitochondria and mPTP opening, the absence of physiological Ca2+ transients following denervation, which leads to the absence of the dynamic Ca2+ uptake into mitochondria, also triggers mPTP opening and initiates mitochondrial dysfunction. We speculate that there may be a biphasic dependence of mPTP on Ca2+ level or direct response to SR Ca2+ release. The molecular composition of mPTP is still incompletely understood. It is unclear what are the molecular mechanisms underlying the different responses of mitochondria to a consistently elevated intracellular Ca2+ level and to the absence of physiological Ca2+ transients following denervation. Future studies are needed to further understand the molecular mechanism underlying the initial response of mitochondria to denervation in skeletal muscle.While mitochondria take up Catraction , 20, 51,of cells , 40. Undynthesis , 52, 53.he cells , 55. Thiformance . Howeverneration . It has oduction , 24. Whis et al. , in whicell line . Most imn of MCU , 59. Whi2+ transients, other muscle adaptations and changes can occur during the first 24 h after denervation. One possibility is that mitochondrial and/or cellular ROS detoxification mechanisms  may be reduced after denervation, and thus, allow for aberrant cell-wide propagation of otherwise spatially restricted mitochondrial ROS-related events. If bursts of ROS-related mitoflash activity are initiated by low-level increases in ROS, then a breakdown of cellular ROS detoxification mechanisms following denervation could also have a contribution to the observed increase in cell-wide waves of ROS-induced ROS release. Thus, changes to denervated fibers in addition to the loss of cytoplasmic Ca2+ transients and mitochondrial Ca2+ uptake are needed to explain the observed enhancement in mitoflash activity. More studies are needed to further understand the molecular mechanism for early response of skeletal muscle to denervation.While denervation eliminates neuromuscular transmission and myoplasmic Ca2+ uptake, which is known to promote the function of the respiratory chain reaction.One concern about our result is the possibility that the drastic reduction of the mitoflash signal in the denervated muscle fibers following the tetanic field stimulation is due to the cell damage caused by the tetanic field stimulation. We were aware of the study of mitoflash signal on normal skeletal muscle by Wei and colleagues who reported that the mitoflash frequency was significantly increased following a 2-s tetanic stimulation and markedly decreased following prolonged 20-s tetanic stimulation . As a re2+ following a brief electric stimulation [2+ transient also follows a similar time course as the cytosolic Ca2+ transient in skeletal muscle fibers. Our results support the hypothesis that the dynamic levels of Ca2+ inside mitochondria follow the time course of the cytosolic Ca2+ transients initiated by motor neuron activation in the physiological condition and that the dynamic change in mitochondrial Ca2+ level could serve as a sensor to decode the nerve innervation. While Ca2+ overload in mitochondria triggers more ROS production [2+ uptake also play a role for maintaining the integrity of normal mitochondrial function.We previously demonstrated that mitochondria in skeletal muscle take up Camulation . Here, woduction , our res2+-induced mPTP opening, and this Ca2+-induced mPTP opening can be blocked by the application of CsA. In addition, the relative level of CypD is increased in mitochondrial fraction of the denervated skeletal muscle, indicating a CypD-related mPTP opening in denervated muscle [2+ transients and the following mitochondrial Ca2+ uptake play a key role in maintaining the functional integrity of mitochondria through a potential mechanism by regulating the mPTP-related mitochondrial ROS production.The coupling of mitoflash and mPTP opening has been well characterized in both cardiac and skeletal muscle , 35. It d muscle . In lined muscle , 62. Stud muscle , 64. It d muscle , 65. Thu2+ transients in maintaining the functional integrity of mitochondria in skeletal muscle. Due to the ongoing debate of mt-cpYFP sensitivity on ROS and pH [Although mt-cpYFP has been characterized and used as a biomarker of mitochondrial ROS generation , 36, 37,S and pH , we used2+ transient is important for maintaining the normal mitochondrial metabolic function by regulating the mPTP-associated mitochondrial ROS generation. Combining voltage-clamp technique and live skeletal muscle cell imaging study, we showed that the Ca2+ level inside mitochondria of skeletal muscle is synchronized with the dynamic change of cytosolic Ca2+ level under physiological condition. In the case of denervation, there are no Ca2+ transients initiated in the cytosol and thus no dynamic alterations in mitochondrial Ca2+ level. Our study suggests that the dynamic change of Ca2+ level inside mitochondria regulates the mitochondrial ROS generation as a response to the cytosolic Ca2+ transient in the physiological condition. Additionally, mitochondria in denervated muscle fibers could sense the absence of the physiological Ca2+ transients and respond with enhanced ROS generation that could initiate downstream signaling toward mitochondrial dysfunction. Future studies should focus on the detailed molecular basis underlying the mitochondrial response to the absence of physiological Ca2+ transients.In summary, we have taken advantage of the transgenic mt-cpYFP mice that allowed us to evaluate the early dynamic response of skeletal muscle to denervation at the mitochondrial level of live muscle fibers. For the first time, our study reveals that physiological CaAdditional file 1: Figure S1.Live cell imaging shows the mitochondrial targeting of mt-cpYFP in skeletal muscle of the transgenic mice (cpYFP). mt-cpYFP targeted well to mitochondria in skeletal muscle of the transgenic mice cpYFP. The representative images of FDB muscle fibers enzyme-isolated from the transgenic mouse cpYFP were incubated with 50 nM TMRE for 10 min. A. Muscle fibers expressing mt-cpYFP. B. TMRE marked mitochondria of the same muscle fibers in A. C. Overlay of A and B indicates the targeting of mt-cpYFP to mitochondria. (PDF 304 kb)Additional file 5: Figure S2.The pure mitochondria and cytosol fractions do not show contamination from each other. The COX-IV antibody only detects bands in pure mitochondrial fraction, not in cytosol fraction, while the GAPDH antibody only detects bands in the cytosol fraction. (PDF 174 kb)


Under Ca2+-free conditions, TG elicited transient Ca2+ increases, which additionally elevated upon the restoration of 2 mM Ca2+. The former were from release of intracellular Ca2+ store and the latter from Ca2+ influx. TG-induced release was inhibited by 2-APB  and chloroquine, and TG-caused influx was inhibited by pyrazole  and stromal interaction molecule (STIM)/Orai channels) and chloroquine. Moreover, chloroquine also blocked Ca2+ increases induced by the engagement of B cell receptor (BCR) with anti-IgM.In this study, thapsigargin (TG), but not caffeine, induced sustained intracellular Ca2+ elevations in splenic B cells through inhibiting Ca2+ permeable IP3R and TRPC3 and/or STIM/Orai channels. These findings suggest that chloroquine would be a potent immunosuppressant.These results indicate that chloroquine inhibits Ca BCR antibodies bind to the BCR, resulting in the phosphorylation of tyrosine in phospholnositide-specific phospholipase C (PLC). The phosphorylated PLC catalyzes phosphatidylinositol-4, 5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). IP3 binds to IP3Rs located on the surface of the endoplasmic reticulum (ER). The activated IP3Rs then mediate Ca2+ release from the ER, leading to increases in intracellular Ca2+ [2+ increases can also be induced by thapsigargin (TG) [2+ increases in B cells remains unclear.Chloroquine is a bitter tastant –4, whichelopment , survivaelopment , activatelopment , and difelopment , 14, cytelopment  and cellelopment . Ca2+ inlar Ca2+ , 16. In gin (TG) . However2+ increases induced by TG and BCR engagement with anti-IgM through inhibiting Ca2+ permeable ion channels.In this study, we found that chloroquine inhibited Ca2+ in primary B lymphocytes from mouse spleens. As shown in Fig. 2+ were induced by TG, an inhibitor of the ER Ca2+ ATPase, which were inhibited by chloroquine. The dose-relationship of inhibition is shown in Fig. 50 was 9.3 ± 0.7 mM , which resulted in transient increases of Ca2+. Ca2+ (2 mM) was then restored in the extracellular solutions, which induced additional increases and were markedly blocked by chloroquine , the incubation of chloroquine abolished TG-induced transient Ca2+ increases and the restoration of 2 mM Ca2+-caused additional elevations . These inhibitions were observed in 129 cells. These results indicate that chloroquine inhibits IP3R-mediated Ca2+ release from the ER.We then studied which Ca of IP3R . As show2+ release from the ER [2+ , which can be inhibited by 2-APB [2+ elevations were mediated by above described ion channels, and which will be inhibited by chloroquine and then resulting in decreases. These results are consistent with previous findings that chloroquine blocks TRPC3 and/or STIM/Orai channels resulting in decreases in Ca2+ levels in smooth muscle cells [+ thymocytes [2+ increases and are blocked by chloroquine. The results show that chloroquine inhibited BCR engagement-induced Ca2+ increases  and light (lights on between 08:00 and 20:00) conditions and were provided adequate water and food. All housing and experiments were performed in according with the Guide for the Institutional Animal Care and Use Committee of the South-Central University for Nationalities.Fura-2 AM was purchased from Invitrogen . Pyrazole-3 (Pyr3) and 2-Aminoethoxydiphenyl borate (2-APB) were purchased from Sigma-Aldrich . Thapsigargin (TG) was purchased from Cayman . RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco . Anti-B220-PE and anti-IgM antibodies were purchased from BD Pharmingen . All of the other chemicals were purchased from Sinopharm Chemical Reagent Co. .l-glutamine. The spleens were then gently teased apart with two G27 syringe needles. The non-cellular tissues were removed by filtering this preparation through a 70-μM nylon mesh. The cells were maintained at room temperature.B lymphocytes were isolated from mouse spleens as previously described . Briefly2+ was measured using fura-2 AM as previously described [2+ levels. The B cells were identified by anti-B220-PE.Intracellular Caescribed , 19. Thet tests were performed to identify significant differences between the means. Differences with p < 0.05 were considered statistically significant.All of the data are presented as the mean ± SEM. The n values represent the number of cells. Unpaired Student’s


In addition, local Ca2+ signals at the endoplasmic reticulum (ER)–mitochondria interface regulate endothelial cell fate by stimulating survival or apoptosis depending on the extent of the mitochondrial Ca2+ increase. The present article aims at describing how remodeling of the endothelial Ca2+ toolkit contributes to establish intrinsic or acquired resistance to standard anti-cancer therapies. The endothelial Ca2+ toolkit undergoes a major alteration in tumor endothelial cells and tumor-associated ECFCs. These include changes in TRPV4 expression and increase in the expression of P2X7 receptors, Piezo2, Stim1, Orai1, TRPC1, TRPC5, Connexin 40 and dysregulation of the ER Ca2+ handling machinery. Additionally, remodeling of the endothelial Ca2+ toolkit could involve nicotinic acetylcholine receptors, gasotransmitters-gated channels, two-pore channels and Na+/H+ exchanger. Targeting the endothelial Ca2+ toolkit could represent an alternative adjuvant therapy to circumvent patients’ resistance to current anti-cancer treatments.Intracellular Ca Herein, Stim1 interacts with and gates the Ca2+-permeable channel, Orai1, thereby triggering the so-called store-operated Ca2+ entry (SOCE), the most important Ca2+ entry route in endothelial cells i in human umbilical vein endothelial cells (HUVECs), which consists in an initial InsP3-dependent Ca2+ peak followed by a plateau phase of intermediate amplitude due to SOCE activation i induced by VEGF in human dermal microvascular endothelial cells (HDMECs) i ,43,44,45s (MAMs) . This corogenase ,48,49. T stimuli ,47,50. F cytosol ,51,52,53ytokines . These iytokines ,54, whicytokines . Growth +i ,43,44ol (DAG) . The folal cells ,57,58,59b-family . More spb-family , whose Cby Orai1 ,63. Moreal cells ,65. Thisal cells  and in re of NCX . Moreove(HDMECs) . Finallyn HDMECs . These dte TRPV4 ,70; NO, te TRPV4 ; adenositivation ; and cyctivation . Moreoveeceptors , acetylceceptors , and N-meceptors . Finallyas TRPP2 , heteromas TRPP2 , TRPV4 i i ,156,159, [Ca2+]i . NO prom [Ca2+]i ,162, inc [Ca2+]i . The admn B-TECs . These earginine , which i(l-NAME) ,164; morsylation  and is psylation . Finallyn HDMECs . Besidesn HDMECs , some ofn HDMECs ,165. Unf2S has also been shown to promote angiogenesis in a Ca2+-dependent manner. For instance, H2S mediated VEGF-induced Ea.hy926 cell proliferation and migration by inducing InsP3-dependent ER Ca2+ release without the contribution of extracellular Ca2+ entry i in ECFCs i. However, NHE-1 blockers, including cariporide and the more specific 3-methyl-4-flouro analog of 5-aryl-4(4-(5-methyl-14-imidazol-4-yl) piperidin-1-yl)pyrimidine (Compound 9t), have been put forward as alternative anti-cancer drugs ER) in RCC-ECFCs, as monitored by using an ER-targeted aequorin Ca2+ indicator ER prevented VEGF from triggering robust intracellular Ca2+ oscillations, proliferation and tube formation, although the pattern of InsP3R expression remained unchanged [2+]ER observed in several types of tumor-associated ECFCs, including IH-ECFCs ER ER could be augmented to such an extent to induce the pro-apoptotic InsP3-driven ER-to-mitochondria Ca2+ transfer. Pinton’s group demonstrated that phototherapy induces a p53-dependent increase in [Ca2+]mit, which leads to tumor disruption in vivo ER is also decreased in the endothelium of tumor neovessels, as ECFCs are likely to be replaced/diluted by local endothelial cells after the angiogenic switch [2+ release in all stromal cells, including T-ECs.Finally, the endothelial Ca Pico145 , 3,5,7-t Pico145 , 2-amino Pico145 , ML204 [ Pico145 , and neu Pico145 . Alterna in vivo ,267. Morn (PMSA) . PMSA isn (PMSA) ,270, incn (PMSA) ,271. Thin (PMSA) . We do n2+. It should, however, be pointed out that several inhibitors of voltage-gated Ca2+ channels, such as verapamil, nifedipine and nitrendipin, are routinely employed in clinical practice to treat severe cardiovascular disorders, including hypertension, arrhythmia, acute myocardial infarction-induced heart failure and chronic stable angina [2+ electroporation on cutaneous metastases of solid tumors as compared to standard electrochemotherapy with bleomycin . Ca2+ electroporation is predicted to enhance the rate of cancer cell death by resulting in cytotoxic Ca2+ accumulation in the cytosol and in mitochondria [2+ signaling did not elicit any intolerable side effects, such as immune depression, bleeding or neuropathic disorders, in least three distinct models of human cancer xenografts [As outlined elsewhere ,32,135, e angina . In agrechondria . Finallynografts ,205,274.2+ toolkit (or transportome) could contribute to the resistance to anti-cancer treatments, which hampers from the very beginning their therapeutic outcome (intrinsic resistance) or leads to tumor relapse (acquired resistance) and patients’ death. The intimate relationship between endothelial Ca2+ signaling and refractoriness to anti-cancer treatments cannot be fully appreciated by studying normal/healthy endothelial cells [2+ signals in normal endothelial cells [2+ toolkit must be first probed on tumor-associated endothelial cells and ECFCs in vitro, as the their Ca2+ machinery could be different from that of naïve cells. The protocol to isolate ECFCs from peripheral blood does not require an unreasonable volume of blood (≈40 mL), but it takes no less than three weeks [2+ toolkit involved in the refractoriness to anti-cancer therapies. Most of the attention is, of course, currently paid to Stim1 and Orai1 [2+ transportome deserve careful investigation. For instance, Orai3 was found to up-regulated in several types of T-ECFCs [2+ entry that regulates proliferation and tube formation in healthy endothelial cells [N-methyl-d-aspartate receptors (NMDARs) are widely expressed in brain microvascular endothelial cells [2+ entry could engage Ca2+-dependent decoders other than eNOS in brain endothelium. Therefore, the expression and role of endothelial NMDARs in glioblastoma should be carefully evaluated.The present article discussed how remodeling of the endothelial Caal cells . For insal cells ,58,251 aee weeks  due to lee weeks . Furthernd Orai1 ,131 and nd Orai1 ,277. Add T-ECFCs ,165, but T-ECFCs . NCX proal cells ,279, butal cells , in whical cells . Aberranal cells  and NMDA


We and others have demonstrated in murine models that CXCR4 inhibitors can mobilize AML cells from the BM into the peripheral blood and enhance the anti-leukemic effects of chemotherapy.3 We have also demonstrated in a previous phase 1/2 study that plerixafor, a small molecule inhibitor of CXCR4, can be safely combined with chemotherapy in patients with relapsed or refractory AML with encouraging response rates.4The interaction of acute myeloid leukemia (AML) blasts with the bone marrow (BM) microenvironment provides potent protection against both spontaneous apoptosis and chemotherapy.5 For mobilization of autologous HSCs, G-CSF acts synergistically when combined with plerixafor and mobilizes higher numbers of CD34+ cells compared with either agent alone.7 In AML, ‘priming' with G-CSF concurrent with chemotherapy may result in superior outcomes for patients receiving induction therapy for AML.8 We hypothesized that disruption of the interaction between leukemic blasts with the marrow microenvironment using G-CSF in combination with plerixafor would effectively mobilize and sensitize AML blasts to chemotherapy.Mobilization of HSCs by granulocyte-colony stimulating factor (G-CSF) occurs through downregulation of mRNA and protein levels of CXCL12, the ligand for CXCR4.3/mm3, acute promyelocytic leukemia, active central nervous system leukemia or who had been previously treated with MEC chemotherapy were excluded from the study. The primary endpoint in phase I was to determine the maximum tolerated dose and in phase II was to determine the complete response rate (CR+CRi) of plerixafor plus G-CSF in combination with MEC in patients with relapsed or refractory AML. The phase I was performed using a standard 3+3 design escalating to a maximum plerixafor dose of 0.75 mg/kg/day. For the phase II, a bivariate design was used in two stages to allow an interim analysis of toxicity and response rates.9 Treatment consisted of G-CSF 10 mcg/kg by subcutaneous injection daily on days 1–8. Plerixafor was administered intravenously (IV) on days 3–8. Chemotherapy consisting of mitoxantrone 8 mg/m2/day IV, etoposide 100 mg/m2/day IV and cytarabine 1000 mg/m2/day (MEC) was administered on days 4–8, ~4 h after administration of plerixafor.In this phase 1/2 study , we evaluated the combination of G-CSF and plerixafor in conjunction with mitoxantrone, etoposide and cytarabine (MEC). Eligible participants were adults, age 18–70 years old, with relapsed or refractory AML. Subjects with a peripheral blood blast count of ⩾20 × 10n=21, 60%) with 10 patients (29%) having had a prior allogeneic hematopoietic cell transplantation , which is less than the 7 out of 20 responses (35%) indicated in the study design for proceeding with the second stage of the phase II. As a result, the study was terminated for futility.Thirty-five patients with a median age of 56 years (range 29–70) were enrolled and treated on this study. The majority received treatment for first relapse (antation . In the 3) was 40 days (range 23–62) from the start of treatment (36 days from MEC). The median time to platelet recovery (platelets ⩾100 K/mm3) was 32 days (range 30–62). Adverse events were typical of those observed for patients with relapsed or refractory AML with the most common non-hematologic adverse events of nausea (69%), vomiting (37%), febrile neutropenia (57%), headache (40%), fatigue (34%), fever (29%) and electrolyte abnormalities  . The median time to neutrophil recovery (ANC⩾1000/mmmia 31%) .To determine the effect of G-CSF and plerixafor on leukemic cell mobilization, peripheral blood samples were collected at baseline, after G-CSF only on day 3 (pre-P), and at 2, 4, 6 and 24 h after IV plerixafor administration . We were4 we observed a decrease in 12G5 binding on AML blasts from pretreatment to 6 h and an increase from 6 to 24 h toward baseline, indicating transient CXCR4 blockade by plerixafor in vivo  and integrin alpha 6 (CD49f) on AML blasts by G-CSF  addition of G-CSF, which downregulates CXCL12 expression and acts synergistically with plerixafor in normal stem cell mobilization; (ii) intravenous instead of subcutaneous dosing of plerixafor to improve the kinetics of administration; and (iii) dose escalation of plerixafor to maximize CXCR4 blockade. Although we demonstrated the safety and feasibility of combining G-CSF and plerixafor with chemotherapy, combination therapy did not improve remission rates compared with historical controls.We believe that primarily two factors contributed to the failure of G-CSF to enhance the efficacy of plerixafor in combination with MEC salvage chemotherapy in our clinical trial. First, a high proportion of patients with unfavorable prognosis were recruited to the study in comparison with our previous study, which was conducted at a single institution . Second,4 We hypothesize that inhibition of CXCR4-mediated prosurvival signaling is more important than the physical detachment and mobilization of AML blasts in enhancing the efficacy of MEC chemotherapy.In our previous study of plerixafor with MEC, blast cell mobilization was not associated with remission.11 As high CXCR4 expression is a marker of poor prognosis in AML, plerixafor-mediated upregulation of CXCR4 may undermine the intended anti-apoptotic effect of CXCR4 blockade by also enhancing the re-homing of AML blasts to the BM, thus reducing the efficacy of CXCR4 inhibitor-based chemosensitization. A number of other CXCR4 inhibitors have been developed for clinical use including other small molecule inhibitors, antibodies and peptidomimetics. Compared with plerixafor, many of these agents provide more potent and sustained inhibition of CXCR4.14 These newer inhibitors also can directly induce apoptosis of AML and other tumor cells line in vitro which supports the assertion that antileukemic activity of CXCR4 inhibitors may be in part independent of the mobilization.15 We are currently exploring the use of these alternative CXCR4 inhibitors, as well as inhibitors of other pathways, which mediate tumor–stromal interactions in both AML and other hematologic malignancies.Plerixafor has a relatively short half-life of 4–5 h and is known to be a weak partial agonist of CXCR4.


Ca2+ dynamics range from global elevations lasting multiple seconds encompassing the soma up to the finest processes, to short elevations restricted to so-called microdomains within fine processes. Investigations of astrocyte Ca2+ dynamics have particularly benefitted from the development of Genetically-Encoded Calcium Indicators (GECIs). GECI expression can be achieved non-invasively in a cell type-specific manner and it can be genetically targeted to subcellular domains. The GCaMP family, a group of GECIs derived from the green fluorescent protein, has experienced some of the fastest advancements during the past decade. As a consequence we are now facing the challenge of needing to compare published data obtained with different versions of GECIs. With the intention to provide some guidance, here we compared Ca2+ dynamics across scales in awake transgenic mice expressing either the well-established GCaMP3, or the increasingly popular GCaMP6f, specifically in astrocytes. We found that locomotion-induced global Ca2+ elevations in cortical astrocytes displayed only minor kinetic differences and their apparent dynamic ranges for Ca2+ sensing were not different. In contrast, Ca2+ waves in processes and microdomain Ca2+ transients were much more readily detectable with GCaMP6f. Our findings suggest that behavioral state-dependent global astrocyte Ca2+ responses can be studied with either GCaMP3 or GCaMP6f whereas the latter is more appropriate for studies of spatially restricted weak and fast Ca2+ dynamics.In recent years it has become increasingly clear that astrocytes play a much more active role in neural processes than the traditional view of them as supporting cells suggests. Although not electrically excitable, astrocytes exhibit diverse Ca They enable localizing and monitoring activity levels in cell populations. Traditionally, chemical dyes have been employed. The first widely used dye, Fura-2, has been developed as a fast, ratiometric Ca2+ indicator that enables quantitative Ca2+ measurements , GC, GC2+ dy at rest . Similar= 0.007) . If GCaM= 0.859) . Togethe2+ transients, such as in spatially restricted microdomains of fine astrocyte processes  and occurred in an asynchronous, uncoordinated manner  than when imaged using GCaMP3 . Astrocytes also exhibit intermediate, wave-like Ca2+ dynamics along individual processes. Since Ca2+ waves are constituted by a spatial and temporal sequence of short Ca2+ transients at any location along a process  knockout mice, whereas microdomain Ca2+ events are smaller but not less frequent [2+ dynamics, the distance to a perfect correlation of 1 was not significantly different when a Ca2+ unresponsive tdTomato signal was analyzed  compared to the GCaMP3 signal in IP3R2 (-/-) mice  within the interlobe linker of CaM restitutes Ca2+ affinity and favors low fluorescence signals of GCaMP5G in the absence of Ca2+ at physiological pH. The latter property is critical for the extended dynamic range of fluorescence change of this indicator during in vivo experiments [2+ bound CaM-M13 complex to the cpEGFP, thereby controlling solvent access to the chromophore and controlling its protonation state, accounting for the increased Ca2+-dependent fluorescence increase [2+ binding sites [2+ affinity [2+-induced CaM-M13 complex formation [2+ sensitivity of GCaMP6f in cultured hippocampal neurons compared to GCaMP3 is predominantly noticeable with small and short Ca2+ elevations. For example, GCaMP6f fluorescence increases in response to 1–2 action potentials are up to 10 times larger than when imaged with GCaMP3 [2+ elevations depend on α1-adrenergic signaling [2+ transients encompass the entire cell and display a time course (>10 s) consistent with Gq-coupled receptor mediated intracellular Ca2+ release. Therefore, our finding that GCaMP6f and GCaMP3 indicate locomotion-induced Ca2+ elevations equally well is not surprising . astrocyte tdTomato (GLAST-CreER(+/-);R26-lsl-tdTomato(+/-), n = 6 mice (43 cells); GCaMP3;IP3R2(-/-), n = 8 mice (52 cells); GCaMP3, n = 9 mice (61 cells); GCaMP6f, n = 11 mice (82 cells). Comparisons, which were not significantly different from one-way ANOVA followed by Bonferroni correction (n.s.), are highlighted. Red lines represent mean ± SEM.(EPS)Click here for additional data file.S1 FileExcel file containing values of all data points presented in this manuscript and underlying statistical analysis. For explanation of organization of the data file, see (XLSX)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Remodeling of the extracellular matrix in human ovarian cancer can be manifested in increased collagen concentration, changes in alignment within fibrils/fibers and/or up-regulation of different collagen isoforms. We used pixel-based second harmonic generation (SHG) polarization microscopy analyses to probe these molecular changes in human ovarian tissues  by: (i) determination of the  Here, we report our efforts in probing collagen molecular aspects, specifically helical structure, concentration, and isoform distribution in normal stroma, benign tumors, and HGS ovarian tumors. This is important as, while the literature supports such modifications, the reports are highly qualitative, and moreover, specific forms of the alterations and their temporal sequences are not known.–To address these molecular changes, here we use several pixel-based polarization-resolved SHG methods including analysis of excitation and emission polarization as well as circular dichroism (SHG-CD) to probe macro/supramolecular structural aspects that further extend our differentiation capabilities beyond morphological metrics.ex-vivo human ovarian tissues, where we examined normal stromal tissues, benign serous tumors, and stage III and stage IV HGS tumors. Benign lesions are of significant interest as studies have shown that benign neoplastic epithelial tumors can progress into low-grade serous ovarian cancer.,We now apply these methods to 22.1All ovarian tissues were obtained using a University of Wisconsin IRB-approved protocol from consented, deidentified patients undergoing surgical debulking treatment for ovarian cancer or benign gynecological conditions. This study examined normal stroma  mounted in the infinity space.For all analyses, we used fields of view that had approximately two-third collagen coverage to be consistent with other analyses we have performed. The pixel-based analysis is then performed over all pixels corresponding to collagen in the image  tests were used to determine mean separation at the 33.1Representative images of each of the four groups are shown in Collagen concentrations were then measured in each tissue using a Sirius Red detection kit and averaged for each sample group. The results of this assay are shown in 3.2We next compared the SHG linear polarization responses  of the four ovarian classification groups.3.2.1Using pixel-based, linear polarization-resolved analysis, the effective pitch angle for each tissue class was determined by reconstructing the SHG intensity as a function of laser polarization dependence by fitting to the molecular model described in detail by Tilbury et al.The extracted peptide pitch angle of the normal tissues is 48.66 deg and is similar to our previous measured results of tendon and 100% Col I gels of 49 deg and 49.11 deg, respectively.3.2.2We next determined the SHG signal anisotropy within fibers in the four classes of ovarian tissues. Instead of acquiring the anisotropy at one angle as most commonly done,3.3Next, we analyzed the same samples using the SHG-CD method described previously4,in-vitro models.,The etiology of ovarian cancer remains poorly understood, for example, even the primary location, i.e., the ovary itself or the fallopian tubes remains an open topic of research.,–It is known during malignant transformation, the epithelial cells undergo an epithelial to mesenchymal transitions,,in-vitro models) and thus are not highly specific. Due to this cross talk, we have not used them here for further validation.It has been suggested by immunofluorescence imaging that Col III is increased or turned over faster in ovarian cancer.The results of the SHG signal anisotropy and SHG-CD measurements revealed similar conclusions to each other. The collagen in normal stroma displayed the highest anisotropy and SHG-CD values, where these were both similar to those in self-assembled Col I gels and tendon. The lowest responses for each were found in benign tumors and are consistent with either an increase in Col III  or altered alignment in Col I molecules within the fibril. Either would decrease the anisotropy or net chirality within the probed focal volume. Relative to normal tissue, the lower SHG anisotropy values in the HGS tumors may correspond to collagen molecules not being uniformly aligned within the collagen fibrils. Our previous work on ovarian cancer suggests that the collagen is newly synthesized as opposed to remodeled existing collagen,We note that while the SHG anisotropy and SHG-CD measurements probe different aspects of structure , the respective findings are self-consistent in terms of structures of the tissue classes. Both these measurements indicate the benign tumors and high-grade tumors all have less organization than the normal stroma, although the underlying reasons are likely different. For example, the benign tumor and the high-grade tumor responses are consistent with increased Col III and protease activity, respectively. Although more work is needed to further characterize the specific molecular changes in structure, e.g., through mass spectrometry, we find statistically significant changes through these label free SHG metrics.in-vivo applications is the significant polarization scrambling due to optical scattering, which can be further amplified by birefringence in collagenous tissues. Specifically, we have previously shown that SHG polarization signatures can be degraded in as few as two scattering lengths, or about 100 microns.in-vivo applications.,,We note that a potential limitation of extending the SHG polarization methods to Although the data acquired in this study used forward emission detection schemes, a backward collection geometry could be used for the SHG polarization analyses, albeit with lower output signal. This is further possible as most tissues show similar features in the forward and backward directions. A common observation is that fibers detected in the backward direction can appear segmented, where this effect arises from the SHG coherence. The coherence length in the backward direction is shorter than the forward and as a consequence, these segments arise from destructive interference within the focal volume, where the same features are continuous fibers when viewed in the forward direction.5We have shown that pixel-based polarization-resolved SHG imaging methods show great promise in delineating normal ovarian stroma, benign tumors, as well as different stages of HGS ovarian cancer by probing molecular changes in collagen. The linear polarization methods to extract the helical pitch angle and collagen molecular alignment and the SHG-CD to probe net chirality provide new complementary information to our previous work on the fibrillar hierarchy. Interestingly, our current findings indicate that Col III is not increased in HGS tumors, as previously suggested in the literature.