The metazoan endoplasmic reticulum (ER) serves both as a hub for maturation of secreted proteins and as an intracellular calcium storage compartment, facilitating calcium-release-dependent cellular processes. ER calcium depletion robustly activates the unfolded protein response (UPR). However, it is unclear how fluctuations in ER calcium impact organellar proteostasis. Here, we report that calcium selectively affects the dynamics of the abundant metazoan ER Hsp70 chaperone BiP, by enhancing its affinity for ADP. In the calcium-replete ER, ADP rebinding to post-ATP hydrolysis BiP-substrate complexes competes with ATP binding during both spontaneous and co-chaperone-assisted nucleotide exchange, favouring substrate retention. Conversely, in the calcium-depleted ER, relative acceleration of ADP-to-ATP exchange favours substrate release. These findings explain the rapid dissociation of certain substrates from BiP observed in the calcium-depleted ER and suggest a mechanism for tuning ER quality control and coupling UPR activity to signals that mobilise ER calcium in secretory cells. These lribution , but preironment , but as ironment .2+. Release of ER Ca2+ has long been known to correlate with disruption of certain BiP-substrate complexes , is the major perturbation to ER function imposed by low [Ca2+].Against this background, we were intrigued by the behaviour of the abundant Hsp70-type ER chaperone BiP upon depletion of ER Caomplexes . Such diomplexes , which iligomers . As chap2+ depletion on BiP function in cells and in vitro. Here, we report on a molecular basis of ER [Ca2+]-dependent regulation of BiP’s functional dynamics under physiological conditions. Our observations provide a plausible explanation for the rapid dissociation of BiP from substrates noted upon ER Ca2+ depletion and suggest a chain of causality, whereby a primary effect of [Ca2+] on BiP’s chaperone cycle gives rise to a secondary proteostatic challenge in the ER.These considerations led us to revisit the effect of ER Ca2+ depletion on trafficking of a model BiP substrate protein through the secretory pathway to the plasma membrane, we chose the T-cell antigen receptor α (TCRα) chain. In absence of other components of the TCR complex, the luminal part of this orphan transmembrane protein interacts with BiP as a substrate and is retained in the ER , generating a reporter (TCRα-N/Q) that was expressed at lower levels (likely due to enhanced degradation) and was more tightly retained intracellularly under basal conditions and conspicuously exposed on the cell surface upon ER Ca release .2+ depletion leads to reporter dissociation from BiP, facilitating ER exit and appearance on the cell surface . Depletion of ER Ca2+ with the ionophore A23187 also led to accumulation of the reporter on the cell surface . We therefore set out to investigate the effect of Ca2+ on BiP function using cell-free approaches.The dissociation of BiP from model substrates emerges as an intriguing feature that distinguishes ER Caind Ca2+ , but a dLike typical Hsp70s, BiP consists of an N-terminal nucleotide binding domain (NBD) and C-terminal substrate binding domain (SBD) connected by a hydrophobic linker peptide . The binQPD, carrying a H-to-Q mutation in the conserved HPD motif of the J-domain  did not significantly alter BiP’s basal ATPase rate but inhibited its JDP-stimulated activity by ∼30%  that is enzymatically coupled to ADP production, confirmed BiP’s known low basal ATPase activity and its stimulation by wild-type JDP cofactor, whereas a non-functional version, JDPJ-domain , did notJ-domain . ReactioJ-domain . Additio by ∼30% . The latactivity .2+  was only slightly affected when up to 1.6 mM CaCl2 was added , with a reduction by ∼51%. This was observed in the context of generally lower ATPase rates measured in the presence of ADP, which directly competes with ATP for binding to BiP. The dependence of Ca2+-mediated inhibition on ADP suggested an important effect of Ca2+ on the nucleotide exchange phase of BiP’s ATPase cycle.We therefore turned to an assay that detects orthophosphate production and allows measurement of ATPase activity in the presence of ADP. The basal ATPase activity of BiP  was similar to values reported earlier (2 (up to 1 mM) had little effect on MABA-ADP release. However, when included alongside the MABA-ADP (and 3 mM MgCl2) during the loading phase, Ca2+ strongly inhibited release in the subsequent dissociation step (decreasing the offk to 0.0171 ± 0.0004 s−1)  yielded a half maximal inhibitory concentration (IC50) of 82.3 ± 16.4 µM (2+ fluctuations in the ER (from >100 µM to low micromolar concentrations) and suggests that this effect could indeed be relevant to BiP function in vivo.Importantly, titrating CaCl 16.4 µM . This va2  and the Grp170-mediated stimulation was enhanced further  for ADP binding to BiP in the presence of Ca2+ compared to Mg2+ , indicating that the T229A mutation has no major impact on BiP’s nucleotide-binding properties  and that both cations occupy the same or overlapping sites in the NBD  to monitor the JDP-mediated binding of BiP to immobilised substrate on the surface of a sensor. This approach tracks transient BiP-substrate interactions in real-time (2+ (QPD) (2+ ([MgCl2]/[CaCl2]=3), suggesting that Ca2+ competes only weakly with Mg2+ under these conditions  . Althougnditions , trace 22+-containing solutions, BiP dissociation was slow in the presence of ADP and fast in the presence of ATP. When present in the dissociation phase, the combination of Mg2+ and Ca2+ ([MgCl2]/[CaCl2]=3) did not affect the rate of ATP-induced BiP dissociation from sensors . Thus, BiP dissociation is subordinate to nucleotide exchange events and is accelerated by ATP binding. Accordingly, in Mg sensors , trace 3bstrates , trace 5P to BiP , it seem2+ and both ATP and ADP ([ATP]/[ADP]=2), the dissociation of BiP from J-domain- and P15-coated BLI sensors was slower compared to a solution containing only Mg2+ and ATP, consistent with direct competition of ADP with ATP for binding to BiP (2+ ([MgCl2]/[CaCl2]=3) to a solution containing both nucleotides further inhibited dissociation of BiP , the NEF Grp170 enhanced dissociation of BiP from substrate-loaded BLI sensors both in the absence and presence of Ca2+ to a similar extent  followed by two sequential dissociation steps. The BiP-loaded sensors were first introduced into solutions containing ADP and either Mg of Ca2+  and reso2+]  regulate BiP’s chaperone activity by modulating the kinetics of productive nucleotide exchange, and thus the rates of substrate release and entry into the next ATPase cycle. These findings propose a mechanism for the decline in BiP-substrate interactions observed upon ER Ca2+ depletion. All other things being equal, at high [Ca2+]ER (resting state) substrate dissociation is slower. Release of Ca2+ from the ER accelerates ATP binding and substrate dissociation, whereas the rate at which ATP hydrolysis-dependent substrate interactions are formed remains largely unaffected. The acceleration of productive nucleotide exchange and substrate dissociation, relative to substrate binding, partitions BiP from its substrates.These observations suggest that changes in /[ADP]=2). Further addition of Ca2+ had the strongest inhibitory effect on the dissociation of mammalian BiP (∼43% decreased dissociation rate) and Drosophila BiP (∼53% decrease), whereas dissociation of the other chaperones was less affected  of the nucleotide as well as indirect contacts to protein residues via several water molecules •Mg2+•ADP structure coordinates only the β-phosphate of ADP directly apo and the NBD of BiPapo containing both its NBD and SBD  and the β-phosphate of ADP (2+ is located between the β- and γ-phosphate groups of ATP (2+ ion in a structure of human Hsp70(NBD) in complex with Ca2+, ADP, and Pi occupies the same position as Mg2+ in a post-hydrolysis state NBD structure  in cycles of ADP rebinding.To establish if the selective effect of Caree NBDs . In eachree NBDs  and the ree NBDs . Overlayolecules , inset. to CaADP . In cont and SBD . The locs of ATP . The Ca2tructure . Togethe2+ on BiP function compared to the other Hsp70s. However, the IC50 values for Ca2+-dependent inhibition of MABA-ADP release from the NBDs of Hsp70 (444.7 ± 80.5 µM) and Hsc70 (345.5 ± 71.1 µM) were much higher than from the NBD of BiP (57.1 ± 7.4 µM)  (52.4 ± 8.1 µM) (50 for inhibition of MABA-ADP release from DnaK(NBD) by Ca2+ was so high (>8 mM) that saturation was not reached over the titration range , which had a high IC50 of 888.0 ± 233.6 µM (2+ seems specific to metazoan BiP.The highly similar CaADP binding mode does not explain the stronger effect of Ca 7.4 µM) . The lat 8.1 µM) . In conton range . Given t233.6 µM . Thus, r2+ ion that is coordinated by the carboxyl group of D257 and backbone carbonyl of H252 in BiP , suggesting low affinity for the cation, and secondly, by the likelihood that this site is accessible to Ca2+ also when the BiP NBD is occupied with MgADP — circumstances under which Ca2+ does not retard nucleotide exchange  measurements revealed that binding of ADP to all NBDs substantially elevated their melting temperatures (Tm), as observed previously for full-length BiP , compared to the cytosolic Hsp70(NBD)s. These findings suggest that the larger decrease in structural flexibility imparted by CaADP binding to the NBD of ER-localised Hsp70s contributes to the relatively stronger inhibition of ADP release by Ca2+.Despite their nearly identical CaADP-bound structures, BiP(NBD), Hsp70(NBD), and Hsc70(NBD) share only 70% sequence identity. We speculated that structural variation that is inapparent in the low-energy, crystallised state may nonetheless lead to functionally important differences in thermodynamic stabilisation by Cangth BiP . The Tm  of Ca2+ . ImportaThe activity of BiP in the ER is subject to post-translational regulatory mechanisms, one of which is the transient sequestration of substrate-free BiP into inactive oligomeric assemblies. These homomeric BiP complexes are based on typical substrate interactions between the SBD of one protomer and the interdomain linker of another protomer . As a coThe strong propensity of BiP to form oligomers and their architecture is further supported by structural data: despite being C-terminally truncated beyond helix A of the flexible SBD lid structure and containing a substrate binding-weakening V461F mutation, we obtained crystals of BiP in multi-component mixtures formed by substrate interactions between the SBD and the interdomain linker of adjacent molecules in the crystal lattice .2+ depletion seems to contradict this function and remained unexplained so far -end would render them refractory to the destabilising effect of ATP. We therefore took advantage of the protease subtilisin A (SubA) that cleaves BiP site-specifically at the linker sequence . Under c230; a signal vastly dominated by BiP). The peptide co-eluted with BiP monomers and oligomers -end]. As expected, addition of ATP to the sample shortly before injection into the SEC column converted BiP oligomers largely into monomers, releasing the bound substrate peptide. As noted previously, treatment with SubA prior to SEC shifted the oligomer peaks to later elution times and abolished the monomer signal in both the A230 and fluorescence channel. However, in contrast to the intact oligomers, the cleaved species were completely insensitive to ATP-induced disassembly and peptide release (We next formed complexes between BiP and fluorescently-labelled substrate peptide by extended incubation of BiP (at concentrations at which it is largely oligomeric) with trace amounts of the peptide. The samples were then analysed by SEC and substrate peptide fluorescence was detected separately from bulk protein absorbance at 230 nm  dissociated from stabilised (SubA-cleaved) BiP oligomers and untreated BiP at similar rates (2+ ([MgCl2]/[CaCl2]=3) had a similar inhibitory effect on MABA-ADP dissociation from both samples. In contrast, under these conditions, Grp170 enhanced the rate of MABA-ADP release from stabilised oligomers only ∼2.9-fold, whereas release from untreated BiP was enhanced ∼12.3-fold  that passive nucleotide exchange occurs with normal rates on BiP oligomers but their conformationally trapped state renders them non-responsive to ATP binding, and (ii) that oligomers are largely resistant to Grp170-stimulated nucleotide release. Together with the finding that BiP oligomers disassemble from one end, these observations suggest a mechanism whereby the relative inertness of the internal protomers kinetically favours BiP oligomers over other BiP-substrate interactions in the Ca2+ fluctuations within the physiological range suggests a defined mechanism that links changes in [Ca2+]ER to protein homeostasis. In the Ca2+-depleted ER, BiP-substrate interactions are destabilised  improves fitness.At high CaADP concentrations, the crystallised NBDs of all Hsp70’s tested bind the cation-nucleotide complex indistinguishably. Solution-based assays suggest that subtle differences in protein dynamics, arising from the 30% sequence divergence between the NBD of metazoan BiP and its cytosolic counterparts, may account for their different sensitivity to Ca2+ release from the ER. The destabilising effect of ER Ca2+ depletion on BiP-substrate complexes may have arisen as a response to situations in which the demands for bulk protein export from the ER (to replenish secreted protein stores) outweighs the benefits of stringent quality control (that would otherwise be imposed by more prolonged BiP-substrate interactions and ER retention). ER quality control responds continuously to the thermodynamic instability of its substrates  is regulated by Cabstrates . The dis2+ depletion. Disagreements on the details notwithstanding, most models for UPR regulation acknowledge the importance of BiP-substrate interactions .Chaperones, in particular BiP, are the most abundant ATPases in the ER . Increas2+ depletion might enhance BiP’s ATPase activity. The cost of this to the ER energy balance may be compensated (at least transiently) by an increase in ATP import into the organelle  origin was confirmed by genomic sequencing. The cells were grown on cell culture dishes or multi-well plates (Corning) at 37°C and 5% CO2 in Nutrient mixture F-12 Ham (Sigma-Aldrich) supplemented with 10% (v/v) serum , 1 × penicillin–streptomycin (Sigma-Aldrich), and 2 mM L-glutamine (Sigma-Aldrich). Cell lines were randomly tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza). All experiments were performed at cell densities of 60–90% confluence. Where indicated, cells were treated with 0.5 µM thapsigargin , 2.5 µg/ml tunicamycin , 10 µM A23187 , 3 mM 2-deoxy-D-glucose , 2 µM PERK inhibitor , and 10 µg/ml brefeldin A . All compounds were diluted freshly in pre-warmed medium and applied to the cells by medium exchange.CHO-K1 cells (ATCC CCL-61) were phenotypically validated as proline auxotrophs and their Chinese hamster (CHOP::GFP XBP1::Turquoise (clone S21) has been described previously (6 cells seeded onto 10-cm dishes 24 hr prior to infection). After 8 hr, the viral supernatants were replaced with fresh medium. The cells were split 48 hr later into medium supplemented with 6 μg/ml puromycin . After further 24 hr, the medium was changed to medium supplemented with 8 μg/ml puromycin. The medium was changed every third day until puromycin-resistant colonies were visible. Individual clones were isolated and tested for reporter expression. CHO-K1 TCRα (clone 24) and CHO-K1 TCRα-N/Q (clone 12) were used for all experiments shown.The UPR reporter cell line CHO-K1 eviously . Cell lieviously  with VSV5 – 1 × 107 cells/100 μl. The cells were washed with 1 ml ice-cold TBS solution [TBS + 3% bovine serum albumin (BSA) + 2 mM CaCl2 + 0.1% NaN3] and pelleted as above. The supernatant was removed entirely and cells were suspended in 100 µl of the same TBS solution containing mouse monoclonal ANTI-FLAG M1 antibodies  at a 1:500 dilution. The cells were incubated 30 min on ice while gently mixing every 10 min. After the incubation time, cells were washed twice in 1 ml TBS solution and suspended in TBS solution containing Alexa Fluor 488 Goat anti-mouse IgG  at a 1:750 dilution for 30 min on ice in the dark as above. The cells were pelleted, washed with TBS solution and suspended in 400 µl TBS solution for flow cytometry analysis. For analysis of UPR reporter induction, CHO-K1 S21 cells were treated as indicated, washed with PBS and collected in PBS containing 4 mM ethylenediaminetetraacetic acid (EDTA).To analyse expression of TCRα and TCRα-N/Q reporters on the cell surface, stable CHO-K1 clones (see above) or parental cells were grown on six-well plates to 80–90% confluence and treated as indicated. The cells were then washed with 1 ml PBS and incubated in 0.5 ml PBS + 2 mM EDTA for 5 min at 37°C. Afterwards, the cells were transferred to a 1.2 ml tube  and pelleted for 3 min in a swing-out rotor at 1000 rpm in a tabletop centrifuge . The supernatant was aspirated and cells were suspended in the remaining liquid to 1 × 10Single‐cell fluorescence signals  were analysed by dual‐channel flow cytometry with an LSRFortessa cell analyser (BD Biosciences). GFP fluorescence and Alexa Fluor 488 fluorescence was detected with excitation laser 488 nm and a 530/30 emission filter and Turquoise fluorescence was detected with excitation laser 405 nm and a 450/50 emission filter. Gating for live cells was based on FSC-A/SSC-A and for singlets was based on FSC-W/SSC-A. Data were processed using FlowJo and median reporter analysis was performed using Prism 6.0e (GraphPad). All events displayed were considered for analysis.g at 4°C and lysed in HG lysis buffer  containing protease inhibitors  with 100 U/ml hexokinase  for 10 min on ice. To release BiP from substrates, hexokinase was replaced by 1 mM ATP during lysis. The lysates were cleared for 10 min at 21,000 × g at 4°C and Bio‐Rad protein assay reagent (Bio‐Rad) was used to determine the protein concentrations of lysates. Equal volumes of normalised lysates were incubated for 2 hr at 4°C with 20 µl UltraLink Hydrazine Resin  on which hamster BiP-specific chicken IgY antibodies had been covalently immobilised (g and washed three times (for 5 min) with HG lysis buffer. The proteins were eluted in 2 × SDS sample buffer and denatured for 10 min at 70°C. Equal volumes of the samples were applied to SDS-PAGE and proteins were transferred onto PVDF membranes. The membranes were blocked with 5% (w/v) dried skimmed milk in TBS and incubated with primary antibodies followed by IRDye fluorescently labelled secondary antibodies (LiCor). The membranes were scanned with an Odyssey near-infrared imager (LiCor). Primary antibodies against hamster BiP , purified as in 2) and supplemented with 175 µM ATP. Note: FICD was excluded from the resulting crystals of oligomeric BiP (see below).For crystallisation, BiP and an equimolar amount of human FICDE. coli DnaJ  as well as the wild-type  and H422Q mutant  J-domain of mammalian ERdj6 (amino acids 390–455) were expressed with an AviTag-His6 sequence at the C-terminus in BL21 T7 Express lysY/Iq cells grown in LB medium containing 50 µg/ml kanamycin. The cells were grown at 37°C to OD600 nm 0.5, transferred to 18°C for 20 min, and expression was induced with 0.5 mM IPTG. The cells were incubated for 16 hr at 18°C during which the recombinant proteins were biotinylated on the AviTag to sufficient degree by the endogenous E. coli BirA biotin ligase. Afterwards, the cells were collected by centrifugation and lysed in buffer G  containing protease inhibitors , 1 mM MgCl2, 0.1 mM TCEP, 0.1 mg/ml DNaseI, and 0.02 mg/ml RNaseA using an EmulsiFlex‐C3 high-pressure homogeniser. The lysates were cleared for 30 min at 45,000 × g at 4°C and the supernatants were incubated with 1 ml Ni-NTA agarose beads per 1 l expression culture for 2 hr rotating at 4°C. The beads were then transferred to a 49 ml gravity-flow column, washed with 1 CV buffer I  and with buffer I supplemented sequentially with (i) 1 M NaCl, (ii) 10 mM MgCl2 + 3 mM ATP, or (iii) 0.5 M Tris–HCl pH 8 + 1 mM TCEP (2 CV each), followed by 2 CV buffer I containing 35 mM imidazole. Proteins were eluted in buffer J  and dialysed against TNM  for 16 hr at 4°C and for further 6 hr against fresh TNM. The protein solutions were frozen in aliquots and stored at −80°C for Bio-Layer interferometry experiments.The J-domain of 2 and 2 mM ADP. Crystallisation of NBDs was performed in 96-well sitting drop plates by combining 200 nl protein solution with 200 nl reservoir solution and equilibration at 20°C against 70 µl reservoir solution. Crystallisation of lid-truncated BiP oligomers was achieved by mixing 100 nl of protein solution and 100 nl reservoir solution. Diffraction quality crystals grew in the following solutions: BiP(NBD) — 26% PEG6000, 0.2 M CaCl2, 0.1 M NaOAc pH 5; Hsp70(NBD) — 10% PEG8000, 0.1 M Tris pH 8.4, 0.2 M CaCl2; Hsc70(NBD) — 24% PEG3350, 0.2 M CaOAc; apo BiP oligomer — 20% PEG3350, 0.1 M Bis-Tris Propane pH 7.5, 0.2 M Na3Citrate; ADP-bound BiP oligomer — 20% PEG3350, 0.2 M K3Citrate. Crystals were soaked in cryosolution  and snap frozen in liquid nitrogen. Diffraction data were collected at the Diamond Light Source beamlines i03, i04, and i24  of hamster BiP (UK2039), human Hsp70 (UK2532), and human Hsc70 (UK2533) were adjusted to ∼50 mg/ml. The preparations were supplemented with an additional 7 mM CaClQPD (UK1966) proteins were diluted from concentrated stocks and exposed to ATP and β-NADH to start the reactions. Proteins were used at final concentrations as indicated. The reactions were performed in HK solution  containing 10 mM MgCl2, 0.05% Triton X-100, 1 mM TCEP. The reactions also contained 4 mM ATP, 3 mM Phospho(enol)pyruvic acid , 0.25 mM β-NADH , and pyruvate kinase/lactic dehydrogenase enzyme mix . Where indicated 3 mM CaCl2 was present. The reactions were carried out in a final volume of 110 µl in 384-well microplates  and β-NADH absorbance at 340 nm (A340) was measured over time at 25°C with a Clariostar plate reader (BMG Labtech). Linear regression analysis was performed with Prism 8 (GraphPad) and the ATP hydrolysis activity was calculated using the molar extinction coefficient of β-NADH (ε = 6220 M−1 cm−1).BiP (UK173), J (UK1965), and J2 and 3 mM ATP in a final volume of 20 µl. Where indicated ADP and CaCl2 was added. The samples were incubated for 1.5 hr at 26°C. Afterwards, 15 µl of each sample were diluted with 135 µl water on a 96-well plate, mixed with 50 µl MG reaction solution, and incubated for 2 min at room temperature. Twenty µl of a 34% sodium citrate solution were added to each well. After further 30 min incubation at room temperature, the absorbance at 623 nm (A623) was measured with a plate reader . Standard curves from serial dilutions of KH2PO4 served as a reference to calculate the specific ATPase activity.Malachite green (MG) reaction solution was prepared freshly by combining stock solutions of MG dye , 7.5% ammonium molybdate, and 11% Tween 20 in a 5:1.25:0.1 ratio. Samples of BiP protein (UK173) at 5 µM were prepared in HK solution containing 3 mM MgClNucleotide release and binding measurements were performed using the fluorescent nucleotide analogues MABA-ADP  and MABA-ATP  carrying a MANT moiety whose fluorescence increases upon binding to Hsp70s . The mea2 and CaCl2 where indicated . For Ca2+ titration experiments, CaCl2 was present in solution A and final [CaCl2] are plotted on the graphs and were used to calculate IC50 values. The dissociation rate constants (offk) were determined by fitting the data to a one phase exponential function using Prism 8.4 (GraphPad). In experiments involving cytoplasmic Hsp70s, all solutions contained 0.2 mM TCEP . Solution B contained assay buffer with 18 mM MgATP. Where specified, 15.6 µM Grp170 were added to solution B. The final concentrations after mixing (release phase) were 1.3 µM for each protein, 1.3 µM fluorescent nucleotides, and 1.5 mM MgATP. The solutions contained MgCl mM TCEP .T229A (UK838) proteins (in solution A) with MABA-ADP or MABA-ATP (in solution B) at the start of the measurement. Solutions A and B contained either 1 mM MgCl2 or CaCl2. Each association rate was calculated from six individual measurements, covering a final protein concentration range from 1.3 µM to 7.8 µM. For that, the data were fitted to a one phase exponential function and the observed rates (kobs) were plotted against the protein concentration to calculate association rate constants (kon) with the equation kobs = kon[protein] + offk using the dissociation rate constants (offk) determined separately under the same conditions.The association rate constants  were detQPD, UK1966) carrying a biotinylated Avi-tag (each at 5 µM in HKTTx) were loaded until a binding signal of ∼0.4 nm was reached. After a brief wash step in assay solution (III) the binding sites on the sensors were saturated with P15 peptide  solubilised at 100 nM in HKTTx solution (IV). The sensors were then washed in HKTTx until a stable baseline signal was established (V). Association of BiP or other Hsp70s was performed in HKTTx solution containing 3 mM MgCl2, 1.5 mM ATP and the indicated proteins at 5 µM (VI). To allow for dissociation, the sensors were transferred to protein-free assay solution with variable components as indicated (VII). The data were processed in Prism 8.4 (GraphPad) and dissociation rate constants were obtained by fitting to a one phase exponential function. When DnaK was analysed  in buffer solution containing 50 mM HEPES-KOH pH 7.4, 100 mM KCl, 0.2 mM TCEP, and 0.05% Triton X-100 (HKTTx). Nucleotide and divalent cations were added as indicated. Streptavidin (SA)‐coated biosensors  were hydrated in HKTTx solution for at least 10 min prior to use. Experiments were conducted at 28°C and 700 rpm shake speed and binding signals were recorded at an acquisition rate of 10 Hz. BLI reactions were prepared in 200 μl volumes in 96‐well microplates . The following assay steps were performed: After an initial equilibration step in HKTTx solution (I), JDPs  and the melting temperature (Tm) was determined as the temperature corresponding to the global minimum of the negative first derivatives of the melt curves.DSF measurements were performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System in 96-well plates  sealed with transparent adhesive film . Samples were prepared in a volume of 25 µl. The solutions contained 50 mM HEPES–KOH pH 7.4, 100 mM KCl, 1 mM TCEP and SYPRO orange protein gel stain at a 1:500 dilution of the manufacturer’s stock . The samples also contained 5 µM NBD protein, 4 mM ADP, and 6 mM MgCl2) in a final volume of 25 µl. The samples were incubated for at least 20 min at 25°C before 10 µl were injected onto a SEC-3 HPLC column  equilibrated with SEC buffer. The runs were performed at a flow rate of 0.3 ml/min at 26°C and peptide bond absorbance at 230 nm (A230) was detected and plotted against the elution time. A gel filtration standard  was applied as a size reference and the elution peaks of Thyroglobulin (670 kDa), γ-globulin (158 kDa), Ovalbumin (44 kDa), and Myoglobulin (17 kDa) are indicated.Samples for analytical SEC were prepared in SEC buffer  calcium depletion activates the unfolded protein response. Here the authors show that calcium selectively affects the dynamics of the chaperone BiP by enhancing its affinity for ADP. These findings explain the rapid dissociation of certain substrates from BiP in a calcium-depleted ER and suggest mechanisms for tuning ER quality control and coupling the unfolded protein response to signals that mobilize ER calcium in secretory cells.Decision letter after peer review:eLife. We apologize sincerely for the delay in getting these comments to you. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Suzanne Pfeffer as the Senior and Reviewing Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Calcium depletion challenges endoplasmic reticulum proteostasis by destabilising BiP-substrate complexes" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Summary:2+ release from the endoplasmic reticulum (ER), either due to signalling processes or induced pharmacologically, triggered the unfolding protein response. However, the link between fluctuations of the Ca2+ concentrations in the ER and protein homeostasis remained unclear. Ron and co-workers investigated the molecular mechanism for this observation with respect to function of the Hsp70 chaperone BiP the main regulator of the unfolding protein response. They found that Ca2+ ion concentrations in the range of physiological fluctuations in the ER influences BiP-protein substrate complexes such that high Ca2+ levels reduce nucleotide exchange rates that are limiting for substrate release. This is a particular feature of metazoan ER Hsp70s and not found to a similar extent for yeast ER Hsp70, for eukaryotic cytosolic Hsp70s and for bacterial DnaK. The authors solved the crystal structure of the nucleotide binding domain of mammalian BiP, cytosolic constitutive Hsc70 and cytosolic heat inducible Hsp70 in complex with ADP and Ca2+ ions and found two Ca2+ ions bound in similar positions in all structures. Comparison of these structures with an earlier solved crystal structure of the nucleotide binding domain in complex with ADP and Mg2+ revealed differences that could explain the increased affinity of CaADP as compared to MgADP. Furthermore, by solving the crystal structure of oligomeric BiP and by isolating and characterizing oligomeric species, the authors elucidated the conundrum that BiP oligomers, though representing a substrate type interaction, was much more refractory to Ca2+-ion depletion than other BiP-substrate complexes. The experiments are conducted very carefully, and the conclusions drawn seem fully justified. Overall this is an excellent study that very elegantly solved a long-standing question in the Hsp70 field and the enigmatic link between Ca2+ signalling and proteostasis in the ER.For many decades it was an intriguing observation that CaBecause of the detailed and thoughtful comments of all the reviewers, their reviews are included for your benefit. With regard to what needs to be done, the reviewers provide this essential summary.Essential revisions:1) To validate the influence of the second Ca-ion in their crystal structure the authors should compare CaADP and MgADP binding to BiP-D257A and BiP-D257N.2) To clarify the influence of the NEF Grp170 on substrate release in the presence and absence of Ca-ions the authors should repeat the BLI assay of Figure 4B conditions 3 and 4 +/- Grp170.2+ on BiP in view of the published record), and then finish with the UPR-relevant experiments, which leave more cell-biological loose ends.3) The structure of the paper might be more logical if it started with the biochemistry  are necessary to slow ADP release for DnaK or yeast BiP. The authors further speculate that increased BiP oligomerization upon Ca2+ depletion is a consequence of the slow dissociation of oligomers, allowing for a redistribution of BiP from BiP-substrate complexes to oligomers.The submitted manuscript aims to address how "fluctuations in ER calcium impact organellar proteostasis." The authors begin by reproducing the previously reported observation that depletion of ER Ca2+ (relative to Mg2+) alters the rates of nucleotide dissociation and association, which impacts the stability of BiP-substrate complexes. Importantly, the impact of Ca2+ in vitro falls within the anticipated range of intracellular calcium fluctuations, suggesting that the in vitro data are relevant to the in vivo secretion of substrate observed upon Tg treatment. Although not a major point in the manuscript, few full-length Hsp70 structures exist in the literature, and the presentation of an additional full-length BiP structure adds also to our knowledge base.The manuscript presents a large amount of work, including a careful biochemical analysis of BiP. The data are convincing that CaWhat remains less fully revealed by the presented work is whether the clean in vitro biochemical observations translate fully to the more complex environment of the ER.2+ and/or Ca2+. Are the differences in rates the authors observe in the presence of Mg2+ vs Ca2+ abrogated by the presence of NEF, which one expects will be present in the ER at the time of Ca2+ depletion? For example, how does the presence of Grp170 alter the relative BiP dissociation from substrate under the various Mg2+/Ca2+/ATP/ADP conditions measured in Figure 4B?1) It would be informative and strengthen the authors' conclusions if they further assessed how the presence of NEF (Grp170) impacts the differences in ADP, ATP, and peptide association/dissociation in the presence of Mg2+ depletion. How do the authors explain this observation in light of their model? This observation from Figure 1 is never specifically revisited in the Discussion; however, the authors speculate that increased protein folding load will lower the lumenal ATP:ADP ratio. Are the authors suggesting that a change in ATP:ADP ratio may be the reason cells pretreated with tunicamycin are more recalcitrant to substrate release from BiP upon Ca2+ depletion? Is PERK inhibition expected to alter the BiP ATPase cycle / alter the ATP:ADP ratio?2) The authors make the interesting observation that increasing the load of folding protein in the ER  limits the secretion of TCR upon subsequence Ca2+ replete and Ca2+ depleted ER. As drawn the model implies a futile cycle role for Ca-ADP and a normal ATPase cycle for Mg-associated nucleotide. One presumes the authors are depicting a Ca2+ replete ER? Showing how the relative flux through the catalytic cycle changes upon depletion in a second scheme would help depict the overall manuscript model. Moreover, do the authors presume there is no Ca-ATP in a Ca2+ replete ER, or just that the Ca-ATP has little role due to a favored associated with Ca-ADP over Ca-ATP? As drawn, the model depicts oligomerization as influenced only by MgATP – in a calcium replete ER, is there a reason why MgATP (versus CaATP) would be the associated nucleotide?3) It might be helpful if the Figure 8 model was split to depict the changes expected for a CaReviewer #3:As I would expect from the Ron lab, there is nothing much to criticize about the quality of the data.2+ in controlling functional properties of BiP. The earlier papers (notably Lodish and Kong) that recorded the remarkable effects of Ca2+ depletion on proteins that depend on the secretory pathway can now be interpreted in light of the findings reported here. These findings are supported by a massive amount of data including structural biological approaches and careful kinetic measurements. On the whole the data are complete and well-presented, although I might have ordered the various elements slightly differently: begin with the clear demonstration of the role of Ca2+ in controlling BiP function, then focus on the data now contained in Figure 1. In this reviewer's opinion, it might be better not to confound the issue of Ca2+ depletion with induction of a UPR at the very beginning of the Results section. The UPR-relevant aspect could be introduced perhaps at the very end of the dataset. The authors should make explicit that for the TCRa N/Q mutant, any impact of applying tunicamycin cannot be attributed to an effect on the reporter itself but must arise from perturbation of glycosylation of other ER-resident proteins, including unidentified proteins that might associate transiently with the reporter, as well as resulting from induction of a UPR. The following (short) section would benefit from a more direct explanation of the rationale for inclusion of tunicamycin: "Next, we examined whether reporter display on the plasma membrane could also arise from an increased burden of misfolded ER proteins, or if it is a feature specifically related to low ER Ca2+.We used TCRα-N/Q for these experiments due to its improved ER retention characteristics. Depletion of ER Ca2+ with the ionophore A23187 also led to accumulation of the reporter on the cell surface ."This manuscript addresses the role of CaHow the lack of the TCR partner subunit(s) , notably TCRb affect the overall structure and folding of TCRa should be discussed. The elimination off the 4 N-linked glycosylation sites further compromises the folding status of TCRa.2+ depletion on secretion and the material discussed in paragraph five and further.In my opinion the Introduction can be condensed, perhaps starting with the Kong Lodish and other papers that first reported the impact of Ca2+ release-mediated reporter export.” Awkward.Results: “Ca Essential revisions:1) To validate the influence of the second Ca-ion in their crystal structure the authors should compare CaADP and MgADP binding to BiP-D257A and BiP-D257N.2+ binding site on BiP’s nucleotide binding domain in panel A). Both mutant proteins showed similar but slightly different basal MABA-ADP release rates compared to wild-type BiP. Importantly, however, like the wild-type protein, both BiP-D257A and BiP-D257N showed much slower MABA-ADP dissociation when nucleotide binding was performed in presence of CaCl2. Moreover, in all cases, presence of the nucleotide exchange factor Grp170 in the dissociation phase enhanced MABA-ADP release. These results indicate that the second Ca2+ binding site  does not have a major contribution to the mechanism by which Ca2+ ions affect ADP dissociation from BiP that is studied in this paper.We have introduced the D257A and D257N mutations into mammalian full-length BiP, purified the proteins and performed MABA-ADP release assays. The data are presented in new Figure 6—figure supplement 3B  To clarify the influence of the NEF Grp170 on substrate release in the presence and absence of Ca-ions the authors should repeat the BLI assay of Figure 4B conditions 3 and 4 +/- Grp170.2. The data are presented in the new panel Figure 4C. The basal BiP dissociation rates in absence and presence of CaCl2 were comparable to the ones observed in Figure 4B. Under both conditions, Grp170 accelerated BiP dissociation from substrate-loaded BLI sensors to similar extend . Therefore, the stabilising effect of Ca2+ on BiP-substrate interactions is maintained even when nucleotide exchange is stimulated by the presence of NEF. We discuss these results in the manuscript.As suggested by the reviewers, we have performed the BLI experiments under the conditions 3 and 4 of Figure 4B and included nucleotide exchange factor (NEF) Grp170 during the BiP dissociation phase alongside ATP, ADP, MgCl2 -/+ CaCl3) The structure of the paper might be more logical if it started with the biochemistry (there is a clear reason to pursue the effects of Ca2+ on BiP in view of the published record), and then finish with the UPR-relevant experiments, which leave more cell-biological loose ends.2+ on BiP’s functionality and thus provide the conceptual background for the detailed mechanistic studies that follow.We thank the reviewers for considering this editorial issue and for their suggested improvement. However, after examining the question empirically (in presentations of the data to colleagues) we feel that the narrative benefits from presenting the cell-based experiments first, because these make an important link to the previously-described, but yet unexplained, observations that hint to a significant influence of CaReviewer #1:2+-ions bound to the NBD, one coordinated to α- and β-phosphate and a second coordinated by the carboxyl group of D257 and the backbone carbonyl of H252. The authors discard this observation, as there is no difference between the different Hsp70s. However, the way in which the second Ca2+ ion is coordinated is very peculiar as this is exactly at the hinge where subdomain IIB tilts outward upon binding of a nucleotide exchange factor. In the absence of NEFs the equivalent of D252 forms an electrostatic contact to the backbone carbonyl of the equivalent of H252, in the presence of a NEF this contact is broken. It is conceivable that this Ca2+ ion stabilizes this interaction. It was proposed earlier that the ATP binding cleft opens and closes also in the absence of nucleotide exchange factors to release nucleotide. Thus, the Ca2+ ion at this position could have a profound effect on nucleotide exchange rates. Such a hypothesis could be tested by mutating D257 to alanine or asparagine. The prediction would be that the effect of Ca2+on the exchange rate would be diminished. Such a mutant could be invaluable for further establishing the physiological role of the Ca2+ regulation.The crystal structure of the BiP-NBD with CaADP showed two CaSee response to Essential revisions point 1.Figure 1: the authors should state more clearly how the flow cytometry data are quantified. Was the geometric mean or the arithmetic mean determined? For logarithmically distributed data like fluorescence the geometric mean should be used. Also, were only the events within the gate considered? This should be clearly stated in the figure legend. TCRα seems to be much more expressed than the mutant TCRα-N/Q. Is the latter more subjected to ERAD. The authors should comment on the differences.In calculating the median fluorescent signal of any given population, all the cells were used . The arithmetic mean of the median fluorescent signal of three experiments ± SD is plotted.In the revised manuscript we comment on the differences in expression levels of the glycosylated and non-glycosylated versions of TCRα.Reviewer #2:[…]2+ and/or Ca2+. Are the differences in rates the authors observe in the presence of Mg2+ vs Ca2+ abrogated by the presence of NEF, which one expects will be present in the ER at the time of Ca2+ depletion? For example, how does the presence of Grp170 alter the relative BiP dissociation from substrate under the various Mg2+/Ca2+/ATP/ADP conditions measured in Figure 4B?1) It would be informative and strengthen the authors' conclusions if they further assessed how the presence of NEF (Grp170) impacts the differences in ADP, ATP, and peptide association/dissociation in the presence of MgSee Essential revisions point 2 outlined above.2+ depletion. How do the authors explain this observation in light of their model? This observation from Figure 1 is never specifically revisited in the Discussion; however, the authors speculate that increased protein folding load will lower the lumenal ATP:ADP ratio. Are the authors suggesting that a change in ATP:ADP ratio may be the reason cells pretreated with tunicamycin are more recalcitrant to substrate release from BiP upon Ca2+ depletion? Is PERK inhibition expected to alter the BiP ATPase cycle / alter the ATP:ADP ratio?2) The authors make the interesting observation that increasing the load of folding protein in the ER  limits the secretion of TCR upon subsequence Ca2+ depletion on BiP, such as enhanced export of substrate proteins like the TCRα reporter. To make this point clearer, we have indicated the predicted effect of a change in the ER ATP:ADP ratio on BiP’s chaperone cycle in the new model presented in Figure 8A. See also explanation in the Discussion section.We agree with the reviewer that there might be a link between unfolded protein stress (caused e.g. by tunicamycin treatment) and a decrease in the ATP:ADP ratio, which may counter the effects of ER Ca2+ replete and Ca2+ depleted ER. As drawn the model implies a futile cycle role for Ca-ADP and a normal ATPase cycle for Mg-associated nucleotide. One presumes the authors are depicting a Ca2+ replete ER? Showing how the relative flux through the catalytic cycle changes upon depletion in a second scheme would help depict the overall manuscript model. Moreover, do the authors presume there is no Ca-ATP in a Ca2+ replete ER, or just that the Ca-ATP has little role due to a favored associated with Ca-ADP over Ca-ATP? As drawn, the model depicts oligomerization as influenced only by MgATP – in a calcium replete ER, is there a reason why MgATP (versus CaATP) would be the associated nucleotide?3) It might be helpful if the Figure 8 model was split to depict the changes expected for a Ca2+ levels and the ATP:ADP ratio on nucleotide exchange. To further increase clarity, we present in panel B the distribution of BiP amongst different pools in a Ca2+-replete ER and its observed relative redistribution in a Ca2+-depleted ER as separate cartoons.We thank the reviewer for pointing out the shortcomings of the original model. We have generated new models for Figure 8: Panel A is a refined version of the original model depicting BiP’s chaperone cycle and substrate interaction-stabilising ADP-to-ADP exchange cycles. We now also indicate the influence of changes in both the ER CaReviewer #3:As I would expect from the Ron lab, there is nothing much to criticize about the quality of the data.2+.We used TCRα-N/Q for these experiments due to its improved ER retention characteristics. Depletion of ER Ca2+ with the ionophore A23187 also led to accumulation of the reporter on the cell surface ."This manuscript addresses the role of Ca2+ in controlling functional properties of BiP. The earlier papers (notably Lodish and Kong) that recorded the remarkable effects of Ca2+ depletion on proteins that depend on the secretory pathway can now be interpreted in light of the findings reported here. These findings are supported by a massive amount of data including structural biological approaches and careful kinetic measurements. On the whole the data are complete and well-presented, although I might have ordered the various elements slightly differently: begin with the clear demonstration of the role of Ca2+ in controlling BiP function, then focus on the data now contained in Figure 1. In this reviewer's opinion, it might be better not to confound the issue of Ca2+ depletion with induction of a UPR at the very beginning of the Results section. The UPR-relevant aspect could be introduced perhaps at the very end of the dataset. The authors should make explicit that for the TCRa N/Q mutant, any impact of applying tunicamycin cannot be attributed to an effect on the reporter itself but must arise from perturbation of glycosylation of other ER-resident proteins, including unidentified proteins that might associate transiently with the reporter, as well as resulting from induction of a UPR. The following (short) section would benefit from a more direct explanation of the rationale for inclusion of tunicamycin: "Next, we examined whether reporter display on the plasma membrane could also arise from an increased burden of misfolded ER proteins, or if it is a feature specifically related to low ER CaIn the revised version we clarify the utility of the unglycosylated version of TCRa to the experiments involving the use of tunicamycin. We thank the reviewer for drawing our attention to this point.How the lack of the TCR partner subunit(s) , notably TCRb affect the overall structure and folding of TCRa should be discussed. The elimination off the 4 N-linked glycosylation sites further compromises the folding status of TCRa.In the revised version we emphasise that impact of expression of TCRα as an orphan and its importance to the experimental design.In my opinion the Introduction can be condensed, perhaps starting with the Kong Lodish and other papers that first reported the impact of Ca2+ depletion on secretion and the material discussed in paragraph five and further.We have shortened the front end of the Introduction, as suggested by the reviewer, focussing on calcium.2+ release-mediated reporter export.” Awkward.Results: “CaThe sentence has been changed.


Astrocytes in these three systems exhibit distinct changes in [Ca2+] following induced death of nearby cells. Changes in [Ca2+] appear to result from release of Ca2+ from intracellular organelles, as opposed to influx from the external medium. Salsa6f expressing astrocytes displayed dynamic Ca2+ changes throughout the phagocytic response, including lamellae protrusion, cytosolic signaling during vesicle formation, vesicle maturation, and vesicle tract formation. Our results demonstrate local changes in [Ca2+] are involved in the process of phagocytosis in astrocytes responding to cell corpses and/or debris.The changes in intracellular calcium concentration ([Ca There is growing appreciation of the functional capabilities of astrocytes, as recent studies demonstrate their participation in previously overlooked cellular processes like synapse remodeling, axonal guidance , axonal Drosophila larvae, astrocytes appear to specifically transform into phagocytes that are the primary cell types involved in the pruning and clearance of synaptic and neural debris during metamorphosis  the Ca2+ sensitive dye Fluo4 loaded into primary astrocytes isolated from mouse brain tissue, (2) the Ca2+ sensitive dye Fluo4 in the established astrocyte cell line Ast1, and (3) astrocytes derived from brain cortex of mice endogenously expressing the ratiometric genetically encoded Ca2+ indicator (GECI), Salsa6f  while monitoring changes in  (initiation of the peak) was determined.For Tin vitro cellular model of astrocyte phagocytosis that allows for the acquisition of fluorescence and corresponding phase contrast images over an extended imaging period. Utilization of laser ablation to mimic a brain injury event (photolysis) permits the precise control of timing and location of cell death, and thus allows direct comparison of cells before and after cell injury. Additionally, the amount of damage induced was limited to a single cell, allowing for the observation of varying events occurring within the visible network of cells.Here we describe an 2+ indicator Fluo4, with the genetically encoded Ca2+ indicator GECI Salsa6f, during visualization of intracellular Ca2+ changes in an astrocyte population exposed to laser ablation. 2+ labeled astrocytes: (1) Fluo4 in the established Ast1 astrocyte cell line, (2) Fluo4 in primary astrocytes isolated from mouse cortex, and (3) astrocytes derived from the cortex of mice expressing the Ca2+ sensitive fluorescent GFAP-Cre Salsa6f probe. Images in 2+ changes within resting astrocytes, within frames 1–2, 6–7, and 11–12 in columns 1 and 2. Astrocytes demonstrate spontaneous Ca2+ flashing and waves that propagated through the astrocyte network, visible in time lapse movies prior to laser perturbations in both Salsa6f and Fluo4 labeled astrocytes  control cells where the laser was fired into an area vacant of cells, adjacent to the observed cell (as opposed to on the cell); (2) “attached” cells that share membrane attachments with the laser-killed astrocyte; (3) “networked” cells indirectly connected to the irradiated cell (in contact with cells directly attached to the killed cell); and (4) “isolated” cells neither in contact with the killed cell nor in contact with networked cells  displayed spontaneous transients occurring at random, with no apparent relationship to laser exposure next to cells  for the different cell categories and sources are displayed in box plots to the right of Ca2+ traces in 2+ signal can be seen in response to photolysis, with Ca2+ fluorescence continuing to increase over the observation time. This is especially visible in traces from primary isolated astrocytes labeled with Fluo4. Isolated astrocytes may need additional time for signaling to reach a threshold for a significant increase in fluorescence to be observed.The transient fluorescence response following laser irradiation compared to baseline intensity (prior to laser irradiation) was calculated as a 2+ transients were detected in cells surrounding the photolyzed cell. There were some variations between the different Ca2+ indicators, mainly that Fluo4 labeled cells did not show a significant difference between the attached and networked primary astrocyte populations whereas Salsa6f labeled cells did (Refer to p > 0.01). For Fluo4 cells, no detectable difference in dF/F was detected between attached (average = 0.53) and networked primary astrocytes (average = 0.42). The dF/F in Salsa6f cells decrease with the relative location from the photolyzed cell increased (attached = 0.53 > networked = 0.42 > isolated = 0.32).In both Fluo4 and Salsa6f cells, CaRefer to . When co2+ oscillation in response to neighboring cell death appears to decay exponentially (R2 = 0.90), but varied in length of time of the decay period (T1/2) calculated for two time periods: pre laser control exposure and post laser exposure for each cell category: control, attached, networked, and isolated. Average T1/2 of peaks prior to laser exposure ranged between 2.8 and 5.3 s .The initial Cay period . Figure T1/2) from the peak following photolysis (at time 0) of Salsa6f Ca2+ oscillation was 29 s (n = 42) for the attached cell group, and 18 s (n = 30) for networked cells. When spontaneous Ca2+ transients occurred in control (n = 36) and post-lysis peak in isolated cells (n = 13), the T1/2 was 5 and 12 s, respectively  except for control cells (p = 0.93). A significantly higher post-lysis T1/2 was observed for attached, networked and, isolated categories when compared to pre-laser T1/2 values .The average time for a 50% decrease , similar to the difference between the average distance from ROI to attached and networked cells. To determine if there is a link between the rate Ca2+ signal diminishes and the distance from the cell to the laser-targeted region, we have plotted T1/2 values vs. the distance between the laser ROI and the closest point of the plasma membrane of the responding cell. Data points in T1/2 and distance of the responding astrocyte to the targeted laser position, as determined using Pearson’s product-moment correlation coefficient. Attached cells had a r2 value of 0.033, and networked cells had a r2 value of 0.00039.The positions of attached and networked astrocytes relative to the damaged cell vary due to the nature of the defined categories. The average distance between the ROI and attached cells is 28 μm, while the average distance between the ROI and networked cell averaged 63 μm. 2+ spiking. An example of two astrocytes responding to the photolysis of an attached cell can be observed in In an interesting observation, a small percentage (less than 5%) of Salsa6f labeled astrocytes responded to photolysis of a neighboring cell with additional and frequent Ca2+ was due to a release of Ca2+ from internal cell organelles, or due to an internal influx through the outer cell membrane, Ast1 cells were bathed in commercially available Ca2+ free MEM without supplementation of bovine serum (subsequently referred to as low Ca2+ medium). The cells were bathed in low Ca2+ medium approximately 2 min prior to imaging  compared with the cells in regular DMEM (0.8 ± 0.11), n = 153 and 117 respectively, p < 0.0001. These results suggest that intracellular calcium stores are largely responsible for the cytoplasmic increases and that external Ca2+ concentrations may affect the degree of Ca2+ release. When the data from all cells was combined, cells in low Ca2+ medium had a shorter T1/2 than cells in Ca2+ medium  (2+ had the shortest T1/2 (8 s) when compared to all groups.To determine whether the transients in cytosolic Ca imaging . After cctively) . Isolate2+ medium . In these experiments Ca2+ free medium is achieved by chelation of any remaining Ca2+ in the commercially available low Ca2+ medium. Cell cultures were perfused slowly with Ca2+ free medium for approximately 3 min. Two single cells within a group of five cells were sequentially killed via photolysis. Cytoplasmic Ca2+ transients occurred during both lysis events  images processed and pseudo colored with look up table fire in Fiji/ImageJ software are displayed in row 3. The second processing method displayed in row 4 of 2+ signal changes. A sharp rise in Ca2+ fluorescence in surrounding cells is observed immediately after photolysis of the central cell, as detected by the shift in fluorescence from blue to green in the row of IMD images in 2+ fluorescence drops back toward baseline/pre laser exposure levels, as detected in images taken 1 min post photolysis. By 36 min post-lysis, signal within the Salsa6f photolyzed cell is absent. Lysis of the cell membrane results in the diffusion and dilution of Salsa6f into the surrounding media, resulting in the decrease in signal observed in 2+ with the formation of phagocytic vesicles and tracts, respectively, discussed in detail in subsequent Sections “Ca2+ Localization During Cell Reorientation” and “Vesicle Tract Formation.”Imaging of Salsa6f astrocytes, requires imaging of 2 channels, cytoplasmic Candicator . Figure 2+ fluorescence, other than infrequent spontaneous cytosolic waves/flashing. Photolysis results in the death of a targeted single cell, followed by a reaction from undamaged neighboring cells. The burst in cytosolic Ca2+ that occurs after damage to the target cell is evident by bright green and red pseudo color fluorescence throughout the cell  ratio of fluorescence intensity. Subcellular resolution of Ca2+ signaling during the phagocytic process allows for tracking local Ca2+ events that are tied with morphological changes occurring during phagocytosis. This response includes a repositioning of the responding cell toward the remaining dead cell debris, and the occurrence of cytoplasmic Ca2+ signaling resulting the formation of endocytic vesicles, and tracts (see subsequent section).We examined the process of cell death in the irradiated cell with respect to changes in Salsa6f fluorescence see , and morRefer to . Phase c2+ flux occurs at the cell’s newly formed leading edge, in the direction of the remaining cellular debris (2+ changes or morphology changes prior to laser exposure. Following photolysis of an adjacent cell (ROI), increased ruffling activity occurs forming a leading edge. This can be seen in the phase contrast image and Salsa G/R ratio images. This is visible in magnified images in rows 2 through 5 of 2+ localizes to the newly formed membrane protrusions, with increased fluorescence corresponding to the dynamic regions oriented toward the debris. Cell edges oriented away from the cellular debris  at 4.5 min post photolysis, which dramatically changes with a formation of an oval fluorescent region at 11 min post photolysis. At 15 min post photolysis, Ca2+ fluorescence increases in intensity, and decreases in geometric diameter. The subcellular circular Ca2+ signal continues to constrict in size and increase in fluorescence as a vesicle forms. In the matching phase contrast images the vesicle is visible at 25 min post photolysis  the leading edge displayed in 2+ was observed to localize in regions surrounding endocytic vesicles. 2+ signal profile for the concentric wave as it progresses to vesicle formation. The G/R signal along the blue ROI is plotted for each time point between 4.5 and 31.25 min post photolysis. Two peaks corresponding to the perimeter of the concentric Ca2+ circle begin to form at 8.75 min, and increase in intensity as they move from the periphery toward the center of the blue ROI. The two peaks meet to form a large central peak at 21 min post photolysis, then diminishes as an endocytic vesicle forms. The position of the vesicle is visualized as a small dip at the center of the trace at 28 and 31.25 min. A small amount of fluorescence surrounding the endocytic vesicle remains following vesicle formation. 2+ during vesicle formation and maturation in two different astrocyte populations.Elevated Ca2+. Elevated Ca2+ levels around the tract persist. IMD ratio images resolve small vesicles that continue to migrate to the periphery of the tract and fuse with the central cavity (see white arrows in Some responding astrocyte with high phagocytic activity formed tracts of vesicles devoid of fluorescence at the cell periphery . Multipl2+] within astrocytes: (1) as an early response to nearby cell damage/death; and (2) during subsequent phagocytosis. We have shown that astrocytes from the adult Salsa6f mouse, embryonic E18 mice (Brain Bits), and from an established Ast1 cell line, all respond with a significant increase in intracellular cytosolic Ca2+ in response to a killed or severely damaged nearby astrocyte. This is consistent with perturbations of astrocytes observed in previous publications, including 2+ in the neocortex region mostly composed of astrocytes following laser induced injury in vivo. It is common to observe spontaneous oscillation in Ca2+ signal in astrocytes; these have been published in numerous studies. There are many known external stimuli resulting in elevated cytoplasmic Ca2+ concentrations and both synchronized and random oscillations including gliotransmitters  are likely due to signaling through intercellular gap junctions that directly link the cytoplasm of neighboring cells. The functional role of gap junction in Ca2+ movement between astrocytes has been well documented in previous studies  (T1/2 ranged 25–60 s).This result was unlike the elevation in Ca2+ dynamics are similar for astrocytes from all three sources with the exception that isolated Ast1 cells exhibited a strong Ca2+ oscillation after the killing event, whereas isolated cells from primary cultures had only small Ca2+ transients. The fact that isolated cells respond to the killing event suggests that diffusible elements such as ATP from the killed cell initiates a Ca2+ signaling cascade within isolated cells, possibly via purinergic receptors on the cell surface  and Multiple EGF-Like Domains (MEGF10). Phosphatidylserine recognition has been shown to be a key step during the initiation of phagocytosis  of the oscillation was lower when Ca2+ dF/F peak amplitude was larger. Since the activity of pumps and channels is modulated by the level of cytoplasmic Ca2+, it seems that different mechanisms are leading to the drop in cytoplasmic Ca2+ within our experiments (2+ medium whose transients are larger than in regular medium are likely activating the sodium Ca2+ exchanger (NCX) and mitochondrial Ca2+ uniporter (MCU). Whereas the cells in Ca2+ containing medium may primarily rely on the higher affinity plasma membrane Ca2+ transport ATPase (PMCA) and sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps to return the cell to low cytoplasmic Ca2+ levels.The duration  can be found below: figshare The animal study was reviewed and approved by the University of California, Irvine, Institutional Animal Care and Use Committee. Written informed consent was obtained from the owners for the participation of their animals in this study.NW, VG-G, MB, and MC designed the experiments for this study. NW, VG-G, HL, JN, EK, PD, LS, and CC executed the experiments, data analysis, and statistical testing. SO developed the Salsa6f mouse. AL provided the mouse tissue preparations. LS developed the software for laser microscopy system. LT, JD, MC, and MB provided the critical feedback and direction of project. MB, NW, VG-G, HL, and PD wrote the manuscript. All the authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


All-trans retinoic acid (atRA), one of the first molecularly targeted drugs in oncology, has greatly improved the outcome of a subtype of AML, acute promyelocytic leukemia (APL). In contrast, atRA has so far provided little therapeutic benefit in the much larger group of patients with non-APL AML. Attempts to identify genetically or molecularly defined subgroups of patients that may respond to atRA have not yielded consistent results. Since AML is a stem cell-driven disease, understanding the effectiveness of atRA may require an appreciation of its impact on AML stem cells. Recent studies reported that atRA decreased stemness of AML with an FLT3-ITD mutation, yet increased it in AML1-ETO driven or EVI1-overexpressing AML. This review summarizes the role of atRA in normal hematopoiesis and in AML, focusing on its impact on AML stem cells.Acute myeloid leukemia (AML) is an aggressive, often fatal hematopoietic malignancy.  Until ror, RARA . Even th utility . Since A utility ,16,17. A in HSCs  and LSCsIn order to sustain the life-long renewal of blood cells, hematopoiesis is organized in a hierarchical manner. The apex of this hierarchy is formed by hematopoietic stem cells (HSCs), a rare, mostly quiescent cell type that resides in a specialized niche in the bone marrow (BM) and is able to both self-renew and give rise to proliferatively active, progressively differentiating progenitor cells . MutatioThe transforming events giving rise to an LSC may take place either in an HSC or in a progenitor cell that consequently regains stem cell characteristics ,24,26,29all-trans retinoic acid (atRA). atRA has greatly improved the outcome of acute promyelocytic leukemia (APL), a subtype of AML characterized by expression of an aberrant retinoic acid receptor [Standard treatment for the majority of patients with AML consists of chemotherapy based on cytosine arabinoside (araC) and an anthracycline for induction. Consolidation comprises further chemotherapy, sometimes complemented by HSC transplantation . Howeverreceptor .RARA, RARB, RARG, and RXRA, RXRB, RXRG, respectively, with additional diversification through alternative splicing [atRA, the major biologically active metabolite of vitamin A, plays multiple roles during development and in the adult organism . Converssplicing ,52,53. Tsplicing ,52,53. Rsplicing .+ CD38− cells and progenitor enriched CD34+ CD38+ cells suggested that the RA pathway was down-regulated in HSCs [In vitro treatment with a pan-RAR antagonist increased the numbers of “cobblestone area forming cells-week 8” (CAFCW8) and of cells with the ability to repopulate severe combined immunodeficiency (SCID) mice , both considered as readouts of human HSC activity. Likewise, co-culture of CD34+ CD38− cells with stromal cells maintained their CAFCW8 activity and SRC numbers. These effects were partially counteracted by chemical or genetic inhibition of CYP26, suggesting that stromal cells contributed to HSC maintenance by inactivating RA [−) CD34+ CD38− cells in G0 during culture, and substantially increased their non-obese diabetic (NOD) SCID repopulating frequency [atRA plays several well-established roles in hematopoiesis, among them the promotion of granulocytic differentiation of committed progenitor cells ,55–57. I in HSCs . In vitrating RA . In a rerequency . Furtherrequency ,61. Howein vitro exposure of HSC enriched lin− Sca1+ c-Kit+ (LSK) cells to the physiological agent atRA enhanced their proliferation and maintained a more immature cell surface marker profile, prolonging their ability to form immature hematopoietic colonies in semisolid media [In vitro experiments after experimental expression of RAR isoforms, as well as competitive repopulation and in vivo limited dilution assays with cells from Rara and Rarg knock-out mice suggested that RARA promoted myeloid differentiation, while RARG mediated HSC maintenance by atRA [In studies using murine HSPCs, id media . Importaid media . The LSKid media . The con by atRA .versus activated HSCs and early myeloid progenitor cells [In vitro and in vivo treatment with atRA enhanced HSC quiescence and serial replating and serial transplantation activity, even under HSC activating stress conditions. By contrast, maintenance of mice on a vitamin A free diet for ~4 months decreased HSC quiescence and activity [Genome-wide gene expression profiling experiments taking advantage of the refined knowledge of the immunophenotypes of murine HSPCs revealed that atRA signaling was highly enriched in dormant HSCs or cells . In vitractivity .ex vs. in vivo exposure – and between the assays used may play a role [Possible explanations for the partially discrepant results regarding the effects of atRA on HSCs include species effects, which may reflect real differences or technical aspects . Also, differences in retinoid treatment – concentration, duration, and y a role ,65. RemaRARA gene. A number of different fusion partners have been described, but about 95% of cases harbor a t, which fuses the promyelocytic leukemia (PML) gene to RARA [Acute promyelocytic leukemia (APL) is a subtype of AML that is characterized by rearrangements of the  to RARA ,46,66. T to RARA ,66. Rema to RARA ,47,66. N to RARA ,66. The  to RARA .via different routes). This differential impact on survival was observed both in the originally treated mice and in secondary recipients transplanted with their BM cells, the latter being considered a readout of leukemia initiating cell (LIC) activity [The molecular and biological mechanisms explaining the success of atRA-based therapies in APL were addressed only after the discovery of their clinical effectiveness. As mentioned above, PML-RARA hinders the functions of both of its fusion partners. It disrupts the formation of nuclear bodies (NBs), subcellular structures in whose genesis the tumor suppressor PML plays a key role, and which regulate multiple cellular functions including proliferation, apoptosis, and senescence ,66. It aactivity . In contactivity . Togetheactivity ,66. In factivity . Thus, aFLT3 internal tandem duplication (ITD) may modulate the response of APL patients to atRA-based therapy [PML-RARA transgene, the additional presence of an FLT3-ITD knock-in allele reduced the effects of atRA on PML-RARA degradation, NB re-formation, granulocytic differentiation, in vivo blast clearance, and on the delay of disease onset in secondary recipients [FLT3-ITD APL, this combination promoted all of the above parameters irrespective of the presence of the FLT3 mutation [FLT3-ITD in the mouse model, an exome sequencing study on matched diagnosis-relapse samples from patients with APL that had been treated with atRA plus chemotherapy showed that FLT3 mutations present at diagnosis were consistently lost at relapse [Even though not undisputed, the presence of specific additional molecular and genetic lesions, in particular, the kinase activating  therapy ,70. In mcipients . In contmutation . Somewha relapse .NPM1, FLT3-ITD, FLT3 tyrosine kinase domain (TKD), MLL partial tandem duplication (PTD), and CEBPA mutations. This analysis suggested that the beneficial effects of atRA were restricted to the  subgroup of patients that had a mutated NPM1 gene but no FLT3-ITD [MN1 experienced improved EFS and OS with atRA [FLT3-ITD, NPM1, or CEBPA mutations, or by MN1 expression [NPM1 mutations [The tremendous success of atRA in APL, together with laboratory observations that atRA promoted differentiation and chemotherapy sensitivity of non-APL AML blasts ,73,74, iFLT3-ITD . In anotith atRA . In contith atRA . This wapression . Also, iutations . A recenutations . It shouutations ,80. It iutations . These rMN1 encodes a transcription co-factor of the RAR/RXR complex, and its elevated expression was associated with atRA resistance in one of the clinical trials [MN1 greatly decreased the sensitivity of a preleukemic BM cell line toward the proliferation inhibiting and differentiation promoting effects of atRA [MN1 in a human myeloid cell line enhanced or repressed the effects of atRA in a gene-specific manner [Complementing the clinical trials, numerous laboratory studies have tried to identify subgroups of AML potentially benefitting from atRA, as well as agents that may sensitize resistant AML cells to retinoids . MN1 encl trials . Accompa of atRA . Inducibc manner .Figure 2NPM1 represent the most frequent recurrent mutation in AML, and lead to a predominantly cytoplasmic localization of the encoded chaperone protein, which usually shuttles between nucleus, nucleolus, and cytoplasm. Building on the reported clinical association between NPM1 mutations and atRA responsiveness [NPM1 mutations were investigated. Both agents, and even more strongly their combination, caused proteasome-dependent down-regulation of mutated, but not wild type, NPM1, leading to re-localization of wild type NPM1  to the nucleus [NPM1-mutated AML cells to respond to atRA- and/or ATO-mediated cell cycle arrest, differentiation, and apoptosis [NPM1-mutated cell line with atRA and/or ATO sensitized it to daunorubicin [NPM1-mutated AML that were deemed unfit for chemotherapy led to transient anti-leukemic effects in three of them [Mutations in siveness , the eff nucleus ,12. Thispoptosis ,12. Furtorubicin . CompassIDH1-mutated cell lines and primary samples were more sensitive to atRA induced differentiation and apoptosis than their IDH1 wild type counterparts, and this was counteracted by an inhibitor of mutated IDH1. In a mouse xenograft model, atRA reduced leukemic burden and increased survival in an IDH1-mutation-specific manner. Most provokingly, pre-treatment with a cell permeable form of 2-hydroxyglutarate, product of the neomorphic IDH1 variant and usually considered an oncometabolite, sensitized AML cell lines with wild-type IDH1 to atRA induced differentiation [Pertinent clinical trials had not been analyzed for an effect of mutations in the genes encoding the tricarboxylic acid cycle enzymes IDH1 and IDH2 on atRA responsiveness, but laboratory experiments suggested a possible relation . IDH1-muntiation .ecotropic viral integration site 1 (EVI1) gene encodes a transcription factor that fulfills essential functions in HSCs, but is down-regulated during normal hematopoietic differentiation [EVI1 expression was up-regulated by atRA in cell lines and in primary AML cells [RARB gene in luciferase reporter assays [EVI1 expression showed that EVI1 enhanced the transcriptional responses to atRA of a number of genes [EVI1 also augmented atRA induced cell cycle arrest, differentiation, and apoptosis in these cell lines [high, but not EVI1low, samples [high AML samples [high AML may specifically benefit from atRA containing therapy, but this assumption was not tested in the relevant clinical trials, and not affirmed by studies on LSCs (see below). However, as another interesting parallel to the situation with APL and with NPM1-mutated AML, ATO targeted the EVI1 protein for degradation via the ubiquitin-proteasome pathway [EVI1 overexpression appeared to confer sensitivity to ATO in murine myeloid cells and in a clinical trial including 28 patients with MDS [The ntiation ,83–85. Intiation . PerhapsML cells ,89–91, dML cells ,91. EVI1of genes . Accordill lines . In prim samples . Preincu samples . This le pathway . Moreovewith MDS .RARB gene, mediated increased DNA methylation and decreased histone acetylation, and prevented induction of RARB by atRA. An shRNA against AML1-ETO or treatment with 5-azacytidine reduced methylation of the RARB regulatory region, and restored the induction of RARB and differentiation in response to atRA. RARB methylation was also found in the majority of primary samples from patients with AML M2 or M4, morphological subtypes of AML often associated with the expression of AML1-ETO or functionally related fusion proteins [AML1-ETO expressing primary AML blasts were found to be atRA sensitive [In contrast to these studies, which identified lesions or gene expression states potentially sensitizing AML cells to atRA, the AML associated transcription factor fusion protein AML1-ETO was reported to confer atRA resistance . In humaproteins . In an iensitive .FLT3-ITD [in vitro experiments led to the suggestion that inhibition of the histone acetyltransferase GCN5 and/or the lysine demethylase LSD1 augmented the anti-leukemic activities of atRA [Some authors raised the question whether the RAR pathway is functional in AML at all, since a number of genes that are mutated or misexpressed in this disease have a negative impact on it . Along tFLT3-ITD . Another of atRA .In vitro experiments with AML cells suggested a role of BM stroma in degrading atRA: human AML cell lines with different driver lesions , as well as primary samples expressing AML1-ETO or related fusion genes, responded to atRA by differentiation and/or loss of clonogenic activity. The activity of atRA was abrogated by co-culture with BM stromal cells, and restored by incubation with a CYP26 inhibitor [CYP26B1 mostly via RARG, RARA-selectivity was considered an additional advantage [NPM1-mutated AML xenograft model [In contrast to the above described efforts, some authors have questioned the availability of atRA in the BM niche. In one study, hematopoietic cells were proposed to reside in a retinoid deplete environment in the BM based on the limited activity of a synthetic reporter gene . In vitrnhibitor . Accordinhibitor . To overdvantage . IRX1951ft model . The RARft model .The above-described studies are mostly based on the concept of “differentiation therapy”, i.e., the assumption that promoting the differentiation of leukemic blasts will effect, or at least support, cure of the disease. However, AML is a stem cell-driven disease and therefore can be cured only by eradication of the  LSC population . Consequ+ cells (identified by Aldefluor staining) than normal controls , while the rest had substantially lower proportions of ALDH+ cells (ALDH-rare AML) [+ and ALDH− cells, gene expression profiling, and xenotransplantation experiments suggested that ALDH-numerous AML contained a higher number of LSCs and these were present among ALDH+ cells [+ cells were enriched for normal HSCs [+ cells were more resistant to araC than ALDH− cells [Both normal and cancer stem cells from a variety of tissues are characterized by high ALDH activity . The humare AML) . The disH+ cells ,105. By mal HSCs . In ALDHH− cells . ConsistH− cells , patientH− cells .et al. investigated the effects of atRA and its interaction with the tyrosine kinase inhibitor sorafenib in AML with FLT3-ITD mutations. atRA enhanced the anti-leukemic effects of sorafenib in AML cell lines, primary samples, and xenografts with an FLT3-ITD [FLT3-ITD allele  with a Nup98-Hoxd13 fusion gene, in vivo treatment with atRA delayed disease onset, and enhanced corresponding effects of sorafenib, in secondary recipients. An in vivo limiting dilution assay with cells from the treated mice revealed LSC frequencies of 1/80, 1/1,700, 1/38,000, and <1/106 for vehicle, atRA, sorafenib, and atRA + sorafenib treated mice, respectively [FLT3-ITD mutation, but also reduced the frequency of LSCs both by itself and together with sorafenib. The ability of atRA to reduce LSC activity in Flt3-ITD driven AML was confirmed in an independent mouse model .While these studies can be interpreted as indirect evidence for a role of atRA in the LSCs of a subset of AML, a small number of studies has directly addressed the impact of atRA on AML stem cells, with heterogeneous results . Ma et aFLT3-ITD . In a coectively . In summFLT3-ITD bearing non-APL AML, the opposite was reported for AML expressing the AML1-ETO fusion or overexpressing EVI1. atRA strongly increased the serial replating ability of AML1-ETO expressing murine BM cells, and resulted in larger, more immature colonies [AML1-ETO-driven AML, the RARA agonist did not prolong survival, but effected a transient decrease in leukemic burden and a persistent increase in myeloid differentiation [While atRA inhibited LSC activity in colonies . Interescolonies . In a contiation .EVI1 and atRA in malignant myeloid cells [MLL-AF9 driven AML was used. MLL-rearrangements are associated with EVI1 overexpression in human AML [Evi1 expression by direct promoter binding – remarkably only in LSK-, but not progenitor-, derived AML [EVI1 overexpression, each observed in about half of the patients, reflects the cell type in which the transforming event occurred also in human MLL rearranged AML [MLL-AF9 driven murine AML [Building on our own previous observations of a functional cooperation between id cells , we askeuman AML ,106,107.ived AML . This sunged AML ,111. Irrrine AML . They werine AML , but arerine AML .Ex vivo treatment of leukemic cells (LCs) from BM of mice with LSK derived, MLL-AF9 driven AML  with atRA augmented the abundance and quiescence of LSCe, and the activity of LSCs as determined by serial replating and in vivo limiting dilution assays [low LCCMP_MLL-AF9, i.e., LCs from mice that developed AML after transplantation with MLL-AF9 transduced common myeloid progenitors (CMPs) [Evi1 in the differential atRA responsiveness of LSCs from LSK- and CMP-derived AML, LCLSK_MLL-AF9 were transduced with shRNAs against Evi1 or with a control shRNA, and transplanted into congenic recipient mice. Experiments with LCs from these mice showed that knock-down of Evi1 per se reduced LSCe/LSC abundance, quiescence, and activity (the first demonstration of a key role of Evi1 in AML LSCs), and additionally abolished the stemness promoting effects of atRA [Evi1 also strongly augmented transcriptional responses of LSCe to atRA: its knock-down reduced the number of atRA-regulated genes to less than one-half the number found in control cells. Pharmacological and genetic inhibition experiments established Notch4, one of the joint targets of EVI1 and atRA, as a relevant mediator of their effects [Ex vivo exposure of BM LCs to a pan-RAR antagonist affected LSCe abundance and quiescence in a manner opposite to that of atRA. Notably, in vivo antagonist treatment significantly prolonged survival of initially treated and secondary recipient mice, and decreased the abundance, quiescence, and activity of LSCe/LSCs. Finally, atRA increased the quiescence of human AML cell lines retaining some stem cell characteristics in an EVI1-dependent manner, and enhanced clonogenicity and LSCe quiescence of primary EVI1 overexpressing, but not EVI1-negative, AML samples [n assays . In conts (CMPs) . To inve of atRA . Evi1 al effects . Ex vivo samples .Evi1, which per se enhanced leukemic stemness, and additionally facilitated the stemness promoting activity of atRA, in AML.Together, these data show that atRA augmented leukemic stemness in AML resulting from HSC, but not progenitor cell, transformation. These differences could be largely explained by differential expression of the stem cell gene FLT3-ITD in combination with a second non-APL AML typical aberration, atRA negatively affected LSC activity [et al., submitted). However, on the background of the APL-typical PML-RARA fusion, the FLT3-ITD counteracted the inhibitory effect of atRA on LIC activity [AML1-ETO-driven mouse model, atRA even promoted LSC properties [MLL-AF9 driven murine AML model in a manner dependent on transformed cell type (LSK cells vs. CMPs) and Evi1 expression [The effects of atRA in the context of AML are multiple and complex. One of its earliest known and best described consequences is the promotion of myeloid differentiation of leukemic blasts, which forms the basis of most studies aiming to identify atRA-susceptible subgroups of patients, and/or substances able to sensitize AML cells to atRA ,97-99. aactivity   resistance and receptor isoform specificity are potentially relevant to the choice of retinoids with optimal anti-leukemic activity ,101. RAR of RARG ,101. Whi of atRA . Thus, tBeyond the choice of the retinoids themselves, the identity of the agents used in combination with them is likely to influence their effects. All clinical studies that investigated the effects of atRA in non-APL AML to date used different protocols, which mThe interest in retinoids for the treatment of non-APL AML is ongoing, as evidenced by numerous recent publications and several currently recruiting clinical trials . Even though the effects of atRA in AML are obviously complex, this research has important potential to lead to improved therapies for additional subgroups of patients with AML.


In this study, we used depolarized MIN6 mouse pancreatic beta cells as a model for evaluating [Cd2+]i detection with commercially available fluorescent probes, most of which have been traditionally used to visualize [Ca2+]i and [Zn2+]i. We trialed a panel of 12 probes including fura-2, FluoZin-3, Leadmium Green, Rhod-5N, indo-1, Fluo-5N, and others. We found that the [Zn2+]i probe FluoZin-3 and the traditional [Ca2+]i probe fura-2 responded most consistently and robustly to [Cd2+]i accumulation mediated by voltage-gated calcium channels. While selective detection of [Cd2+]i by fura-2 required the omission of Ca2+ from extracellular buffers, FluoZin-3 responded to [Cd2+]i similarly in the presence or absence of extracellular Ca2+. Furthermore, we showed that FluoZin-3 and fura-2 can be used together for simultaneous monitoring of [Ca2+]i and [Cd2+]i in the same cells. None of the other fluorophores tested were effective [Cd2+]i detectors in this model.The understanding of cellular Cd Intracellular accumulation can occur through a variety of pathways normally used by Ca2+, Zn2+, and other metals  and are easy to use. The first live cell studies of [Cd2+]i uptake appropriated the iconic Ca2+ probe fura-2, which also has extremely high affinity for Cd2+ , which express much of the machinery important to pancreatic beta cell function, including L-type voltage-gated Ca2+ channels that are readily activated with high extracellular potassium  ethylenediamine (TPEN), and fluorescent probes  were purchased from Thermo Fisher Scientific. Fura-2FF was purchased from TEF Labs. Fetal bovine serum was purchased from Midwest Scientific. All other reagents were purchased from Sigma-Aldrich unless otherwise noted.2. Round glass coverslips  were coated with poly-l-lysine (Sigma #P4707) according to the manufacturer’s recommended protocol, and trypsinized cells were plated at a density of 100,000 cells per coverslip placed in 12-well culture plates. Media was changed every 2 days, and cells were used 5–7 days following plating. Cells from passages 23–42 were used in experiments.The mouse insulinoma cell line (MIN6) was generously provided by Dr. Malek El Muayed at Northwestern University, Feinberg School of Medicine and maintained according to previously published methods . Briefly3, 0.9 MgSO4, 1.4 CaCl2, 20 HEPES, and 5.5 glucose, pH adjusted to 7.4 with NaOH. Depolarization was achieved with a high KCl, phosphate-free buffer containing (in mM): 92 NaCl, 50 KCl, 10 NaHCO3, 0.9 MgSO4, 1.4 CaCl2, 20 HEPES, and 5.5 glucose, pH adjusted to 7.4 with NaOH. For nominally Ca2+- free experiments, CaCl2 was omitted from both the HBSS and high KCl buffer. For loading of dye, cells were incubated for 30 min at room temperature in 500 μL loading buffer comprised of HBSS supplemented with 5 μM acetoxymethyl (AM) form of the fluorescent probe and bovine serum albumin (5 mg/mL). Coverslips with dye-loaded cells were placed in a 500 μL perfusion chamber and mounted on the microscope stage. The chamber was superfused at 5 mL/min with CaCl2, CdCl2, ZnSO4, sodium pyrithione, MnCl2, TPEN, and/or Ca2+ channel blockers at the indicated concentrations.Live-cell fluorescence microscopy was carried out in a HEPES-buffered, phosphate-free salt solution (HBSS) containing (in mM): 137 NaCl, 5 KCl, 10 NaHCOLive-cell microscopy of single dye experiments were performed at room temperature on an IX70 Olympus fluorescence microscope equipped with a 40× oil-immersion objective . Excitation light was produced by a xenon lamp-based Lambda LS stand-alone illuminator with filter wheels (Sutter Instruments). Optics for fura-2, fura-2FF and mag-fura-2 used alternately switched 340/20 and 380/11 nm excitation filters, 400 nm dichromatic mirror, and 510/84 nm emission filter. Optics for indo-1 used 340/10 nm excitation filter, 387 nm dichromatic mirror, and alternately switched 405/30 and 485/25 nm emission filters. For Calcium Green-5N, Fluo-5N, FluoZin-1, FluoZin-2, FluoZin-3, Leadmium Green, and Newport Green DCF: 480/20 nm excitation filter; 500 nm dichromatic mirror; and 520 nm emission filter. For Rhod-5N: 540/20 nm excitation filter; 570 nm dichromatic mirror, and 590 nm emission filter. Images were captured with an LT Flash4.0 CMOS camera with HCImage acquisition software (Hamamatsu Photonics).Dual dye experiments using fura-2 and FluoZin-3 were performed on a Nikon Eclipse TE2000-S fluorescence microscope equipped with a 40× oil-immersion objective , illuminated by a xenon lamp-based Lambda DG-4 high speed filter changer (Sutter Instruments). Cells were incubated in loading buffer supplemented with 5 μM fura-2 and FluoZin-3 for 45 min at room temperature. For fura-2, excitation light was modified by alternately switched 340/11 and 380/11 nm excitation filters. For FluoZin-3, light was modified by a 495/10 nm excitation filter. A 510 nm dichromatic mirror and 560/80 emission filter were used for both dyes. The camera and software were the same types used in the Olympus system described above.2 or ZnSO4 were added in the range of 0–9 mM, to a final well volume of 100 μL. Free metal concentrations were calculated using the Maxchelator program (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/webmaxc/webmaxcS.htm). Data were represented as a percentage of fluorescence at baseline (50 μM TPEN) versus log of free metal (in M). KD values were calculated by curve-fitting the data using nonlinear regression of one-site specific binding with Bmax constrained between 0 and 100.Spectrofluorimetric measurements of the tetrapotassium salt form of FluoZin-3 were performed on a Perkin Elmer Enspire multimode plate reader. Fluorescence was recorded from 96-well black plates with 495 nm excitation and 517 nm emission. Experiments were performed in Chelex 100-treated buffer containing (in mM): 140 KCl, 10 MOPS, 10 nitrilotriacetic acid (NTA), pH 7. FluoZin-3 salt was added at a final concentration of 500 nM, and serial dilutions of metal from 1000 × stock solutions of CdCl2+ levels were measured by inductively coupled plasma mass spectrometry (ICP-MS) in samples sent to the Michigan State University Veterinary Diagnostic Laboratory. MIN6 cells were plated in 12-well plates coated with poly-l-lysine and maintained in growth media for 5–7 days. All HBSS buffers were made nominally Ca2+-free. Cells were rinsed twice in non-depolarizing HBSS, then treated with 0, 30 or 300 μM CdCl2 in 2 mL volume of depolarizing HBSS for 5 minutes. Other wells were treated with 30 μM CdCl2 in non-depolarizing HBSS. Plates were placed on a rocker during the 5-minute treatment to simulate bath perfusion use in the microscopy experiments. Cells were rinsed three times in non-depolarizing HBSS containing 100 μM EDTA to remove any extracellular bound Cd2+, followed by a final rinse in non-depolarizing HBSS. Cells were lysed using 0.1 mL commercially available lysing buffer (Thermo Fisher Scientific 78505). For each trial, cell lysates were pooled from three wells and experiments were repeated in at least three different cell culture passages.Total intracellular Cd0), after background-subtraction. For a given coverslip, fluorescence was typically collected from a field of 30–60 cells. Representative data traces are plotted as an averaged fluorescence change from all cells within a field from one coverslip. Summary data are presented in bar graphs as mean ± SD consisting of four to six coverslips for each condition, using cells from at least three different cell culture preparations. Where appropriate, statistical analyses used Welch’s t-test or ANOVA with Tukey’s post hoc test for multiple comparisons. Statistical significance was determined as p ≤ 0.05.Statistical analyses and graphical representations of data were performed with GraphPad Prism 8.1 software. For the fura dyes, the ratio was calculated as the background-subtracted signal at 340 nm divided by the background-subtracted signal at 380 nm. For indo-1, the ratio was similarly calculated as the background-subtracted signal at 405 nm divided by the background subtracted signal at 485 nm. For single wavelength dyes, fluorescence signal (F) was divided by the starting fluorescence value  when Ca2+ is omitted from the buffer. In contrast to the kinetics of the [Ca2+]i signal, the [Cd2+]i rise was much more gradual and remained elevated after depolarization was finished. Restoration of baseline fluorescence did not occur spontaneously but instead required application of the membrane-permeant, heavy metal chelator TPEN (50 μM). We confirmed that L-type voltage-gated Ca2+ channels (VGCCs) are a major route of accumulation for both ions under these circumstances. The standard L-type blockers nifedipine, nitrendipine, and verapamil inhibited fura-2 responses as much as 89% for [Ca2+]i and 77% for [Cd2+]i . While this tactic helps to minimize signal confounds that are expected when exploiting a high-affinity [Ca2+]i probe for the detection of [Cd2+]i, it diminishes the physiological relevance of these experiments. In 2+]i changes over a range of extracellular Ca2+ concentrations. When extracellular Ca2+ is absent or minimal  stimulated a small but consistent FluoZin-3 increase (about 1.5-fold). This was prevented by co-treatment with TPEN (data not shown), suggesting that Ca2+ influx mobilized a small amount of intracellular Zn2+, which is consistent with previous reports that documented Ca2+-induced Zn2+ mobilization in other cell types  using the ionophore sodium pyrithione  using depolarization , the rate of Mn2+ quench would be slower for a signal dominated by [Cd2+]i. Conversely, if the “[Cd2+]i response” was actually due to [Zn2+]i that was displaced by Cd2+, then the quenching kinetics in the Cd2+ trial would not differ from the Zn2+ trial. Our data show that [Mn2+]i extinguished the 340 nm component of the [Cd2+]i signal at a rate slower than a corresponding [Zn2+]i signal , in cells depolarized in the presence of 50 μM Zn2+ or Cd2+. When considered as a whole, the data in 2+]i loads achieved in our model behave as free Cd2+ that interacts directly with the dye. It remains possible that some Zn2+ is liberated from intracellular binding sites, but this appears to be minimal.The abundance of intracellular Znrithione , or withrization  in orderio units , comparenits , the resulting [Ca2+]i load clearly becomes an important component of the fura-2 signal. Because fura-2 affinity for Cd2+ (KD ~ 10−12M) is about five orders of magnitude higher than for Ca2+ (KD ~10−7M) the ability of [Ca2+]i to interfere with the [Cd2+]i response is perhaps counterintuitive. However, here it is necessary to remember that methods using passive dye loading of AM esters can achieve very high intracellular dye concentrations— hundreds of micromolar, possibly even millimolar. In such conditions, the binding reaction is more a function of reactant concentrations, while the KD becomes less important , could effectively compete for Cd2+ in the presence of fura-2 (KD Cd2+ ~ 10−12 M). This seeming paradox is perhaps explained by competitive binding dynamics, where a surfeit of [Ca2+]i consumes much of the fura-2 binding capacity, leaving [Cd2+]i free to bind with FluoZin-3.The combination of fura-2 and FluoZin-3 offers a potentially useful approach for studying the relationship between [Cd2+]i. Other groups have used Leadmium Green to report Cd2+ uptake in various plant and animal systems (2+]i sensor, so long as any concomitant [Zn2+]i perturbations are kept minimal (2+]i accumulation in the MIN6 cells used in the present study. The inconsistency between our current and previous data might be explained by the different methods used to deliver [Cd2+]i: the previous work used the ionophore pyrithione, which probably introduces a much larger [Cd2+]i burden compared to what can be achieved via activation of endogenous ion channels. Other probes we evaluated, e.g. indo-1, Rhod-5N, mag-fura-2, etc., had shown promise in cell-free systems (2+]i accumulation, none of them proved effective as live cell [Cd2+]i sensors. These results underscore the inherent difficulties in translating in vitro Cd2+ responsiveness into practical utility inside the living cell.In the current study, we were most surprised by the nearly complete failure of Leadmium Green to respond to i probes. FluoZin-3 and fura-2 proved most effective, but care must be taken to assure that their signals are not confounded by [Zn2+]i or [Ca2+]i, respectively. The combination of FluoZin-3 and fura-2 offers a novel method for the simultaneous detection of [Cd2+]i and [Ca2+]i.In this study we have carefully evaluated a collection of commercially available Cd