In order to investigate the physiological role of this interaction, we examined expression and localization of SPCA2 and Orai1 in mouse lactating mammary glands. We observed co-induction and co-immunoprecipitation of both proteins, and isoform-specific differences in the localization of SPCA1 and SPCA2. Three-dimensional cultures of normal mouse mammary epithelial cells were established using lactogenic hormones and basement membrane. The mammospheres displayed elevated Ca2+ influx by store independent mechanisms, consistent with upregulation of both SPCA2 and Orai1. Knockdown of either SPCA2 or Orai1 severely depleted Ca2+ influx and interfered with mammosphere differentiation. We show that SPCA2 is required for plasma membrane trafficking of Orai1 in mouse mammary epithelial cells and that this function can be replaced, at least in part, by a membrane-anchored C-terminal domain of SPCA2. These findings clearly show that SPCA2 and Orai1 function together to regulate Store-independent Ca2+ entry (SICE), which mediates the massive basolateral Ca2+ influx into mammary epithelia to support the large calcium transport requirements for milk secretion.An unconventional interaction between SPCA2, an isoform of the Golgi secretory pathway Ca This signaling function was independent of Golgi and ER Ca2+ stores, and did not require ATPase or transport function of SPCA2. Constitutive elevation of Ca2+ influx in these cells activated MAP kinase signaling pathways and promoted tumor proliferation. Knockdown of either SPCA2 or Orai1 expression was found to attenuate Ca2+-signaling and inhibit tumor growth in mouse xenografts. Analysis of mutants and chimeras revealed that the membrane-anchored C-terminal tail of SPCA2 was sufficient for interaction with Orai1 to elicit Ca2+ influx ATP2C2, encoding a ∼20 kDa membrane-anchored C-terminal domain is expressed in several secretory tissues under control of the helix-loop-helix transcription factor MIST1 We showed that SPCA2, but not SPCA1, traffics to the plasma membrane in the breast cancer derived MCF7 cell line, where it interacts with the store-operated Ca2+ and bound calcium, ranges between 40–80 mM in various mammalian species 2+ transporters, regulators and binding proteins must be upregulated to drive transcytosis of calcium while maintaining submicromolar cytoplasmic Ca2+ concentrations to avoid Ca2+ mediated toxicity and cell death. Transport and sequestration of Ca2+ is achieved by coordinated increase in the expression of Ca2+ pumps, and channels 2+ at the basolateral membrane via yet to be described Ca2+ channel(s) 2+ entering the mammary secretory cells then travels one of 2 routes to secretion into milk. In the first transport route  Ca2+ is likely rapidly pumped in Golgi/secretory stores via SPCA1 and/or SPCA2 where it is bound to casein to facilitate casein micelle formation, packaged in secretory vesicles and secreted into milk primarily as Ca2+-casein 2+ is pumped directly across the mammary secretory cells apical membrane into milk by PMCA2bw 2+ while it is shuttled to PMCA2bw are unknown but calbindin-9k is a candidate calcium binding protein that could provide this function 2+ directly into the secretory vesicles while it is trafficked to the apical membrane Total calcium concentration in milk, including ionized Ca2+ entry into the lactating mammary secretory cells, by interaction with Orai1 channels 2+ influx into mammary epithelial cells Our observations point to a new role for store-independent Ca2+ influx (SICE) in the sequestration of Ca2+ from the blood for transport to milk.The least understood part of mammary calcium transport into milk is the mechanism by which calcium enters the basolateral membrane of the lactating mammary secretory cell. Based on our recent findings in mammary tumor cells, we evaluated a potential role for SPCA2 in eliciting Ca2+ channels, transporters, buffering proteins and regulators, and must be tightly modulated to avoid cytoplasmic calcium toxicity. Previously, analysis of transcripts from mouse mammary gland tissue starting at Day 10 before parturition revealed induction of all three classes of Ca2+-ATPases, including isoforms of SERCA, PMCA and SPCA pumps 2+-ATPase was induced early and significantly prior to parturition. Subsequently, isoform-specific differences in mRNA were noted, with SPCA2 showing higher transcriptional elevation relative to SPCA1 in lactating mouse mammary gland 2+ channel previously described to elicit Ca2+ influx in breast derived tumor cells Lactation is characterized by massive transcellular flux of calcium, from the basolateral side of mammary alveolar epithelium (blood) into lumen (milk). This involves coordinated induction of a host of Ca2+ flux.Previously, Faddy et al. 2+ handling for SPCA2 and Orai1 in a lactation model, we turned to a three-dimensional culture that mimicked some aspects of native lactating tissue while still retaining the advantages of in vitro cell culture. The mouse mammary epithelial line SCp2 responds to basement membrane (Matrigel) and lactogenic hormone (prolactin) by differentiating into alveolus-like structures characterized by induction and secretion of milk protein, β-casein 2+ transport as seen in native tissue. Other Orai and STIM isoforms also showed varying levels of transcriptional induction  upon reintroduction of extracellular Ca2+ previously shown to be necessary and sufficient to interact with Orai1 and elicit Ca2+ imaging. Neither full-length or C-terminal domain of SPCA2 fully restored thapsigargin-sensitive stores .shSPCA2 knockdown and re-transfected SCp2 cells were loaded with Fura-2 for Cae stores , again c2+ entry (SICE) elicited by SPCA2 via interaction with Orai1 (5). Following brief exposure of SCp2 monolayer cells to nominally Ca2+ free medium, reintroduction of extracellular Ca2+ was accompanied by rapid and transient influx that was abolished by knockdown of either SPCA2 or Orai channels , shown in 2+ entry (SICE) that is unmasked upon brief removal of extracellular Ca2+. As additional demonstration of this mechanism of Ca2+ entry, we evaluated SICE in shSPCA2 treated cells that were transfected with full-length or C-terminal domain of hSPCA2. Both constructs were able to confer elevated and sustained Ca2+ entry to the SPCA2 knockdown cells, consistent with plasma membrane delivery and functional rescue of Orai1 -requiring Ca2+ efflux via active sequestration of Ca2+ into the secretory pathway. However, SPCA2 is restricted to polarized cells of secretory or absorptive epithelia  and centrifuged at 8 rpm for 2 minutes to remove excess trypsin. Cell growth was monitored using Cell Titer 96 AQ from Promega according to manufacturer’s instructions. For mammospheres, trypsinized cells were then mixed with a solution containing 50% Matrigel (Sigma) and 50% differentiation media (DMEM/F-12 (1∶1), hydrocortisone (1 µg/ml), insulin (5 µg/ml) and recombinant mouse prolactin (3 µg/ml). Cells were plated at 1×103 on a 100% Matrigel matrix on 25 mm round coverslips. Matrix was again allowed to solidify prior to the addition of differentiation media to cover the layered coverslip. Mammosphere culture was allowed to differentiate for 10 days, changing the media every second day, prior to assays for differentiation.SCp2 cells  3 cells per coverslip. These coverslips were placed in a standard cell culture incubator and allowed to differentiate. Differentiation liquid media was changed every two days and replaced with fresh media. Cellular clusters (consisting of dense groups of >4 cells each) were counted and categorized into one of three categories: (1) undifferentiated clusters- cells which maintained characteristic of monolayer culture but were in close proximity to one another (single cells); (2) spheroids- partially differentiated cell clusters with characteristics which showed lack of tight junction formation and lumen; (3) mature mammospheres- cells which grouped into a differentiated mass with tight junctions and a lumen. All three classifications were considered clusters (belonging to group 1) but the proportion of spheroids to mature mammospheres and the percentage of cells, which went through the entire development program, were noted.SCp2 cells were plated on 25 mm coverslips for three-dimensional culture at 106 cells were washed with sterile Hank’s Balanced Salt Solution  prior to RNA extraction. An RNAeasy kit was used to collect the total RNA via centrifugation (Qiagen) per the manufacturer’s instructions and quantified by absorption at 260 nm by spectrometry. RNA was converted to cDNA by reverse transcription using iScript (Bio-Rad Laboratories). Equal amounts of cDNA was subjected to PCR amplification for specific transcripts using the following primers: Orai1∶5′-ACCCCACGAGCGCATGCATC-3′ (forward) and 5′-GCTTGGTGGGGCTTGGCTGT-3′ (reverse); Orai2∶5′-CTGAGGTGGTCCTGCTCT-3′ (forward) and 5′-GGTAGAAGTGGATGGTGAAG-3′ (reverse); Orai3∶5′-CATCCACAATCTCAACTCTG-3′ (forward) and 5′-ATAGAAGCAGAGGATGGTGT-3′ (reverse); STIM1∶5′-AAGAGTCTACCGAAGCAGAG-3′ (forward) and 5′-GTGCTATGTTTCACTGTTGG-3′ (reverse); STIM2∶5′-GTGCGCTGGGTCGGAAGAC-3′ (forward) and 5′-GGGGCACCAGATCGCATCG-3′ (reverse); SPCA1∶5′-CCAGTGTGGCCGTGGCTGAC-3′ (forward) and 5′-TCAGCCTGGAGAAGGCCTGCAA-3′ (reverse); SPCA2∶5′-GACCTGCTGCTGCTGACGGG-3′ (forward) 5′-CAGGCCAGAGGCACCCAAGC-3′ (reverse); PMCA2∶5′-CAGGGTCTGCCACCCTCGGAG-3′ (forward) and 5′-CATGGTCGGGACAGCTCCCCTA-3′ (reverse); SERCA2B: 5′-ACTTCTTGATCCTCTACGTG-3′ (forward) and 5′-AGACCAGAACATATCGCTAA-3′ (reverse); actin: 5′-GCAGCTCCTTCGTTGCCGGT-3′ (forward) and 5′-TACAGCCCGGGGAGCATCGT-3′ (reverse). The number of amplification cycles was adjusted to ensure that generation of products was not saturated. Loading control was mActin.102 (2 mM), sucrose (300 mM) and a complete protease inhibitor cocktail  at pH 7.5. The homogenate was mixed with an equal volume of Buffer B (Buffer A plus 0.3 M KCl) and centrifuged at 4000 g for 10 min. The supernatant was collected, adjusted to 0.7 M KCl, and centrifuged at 100,000 g for 1 h. The supernatant was discarded and the pellets were resuspended in Buffer C (Buffer A plus 0.15 M KCl).Mammary tissue microsomes were prepared as previously described Microsomes from mammary tissue were incubated for 15 min at room temperature in a modified Laemmli buffer containing 150 mg/ml urea and 65 mM DTT. Samples were then electrophoresed for 50 minutes at 200 volts in a 4–12% Novex NuPAGE® Bis-Tris Gel using MOPS SDS running buffer . Proteins were transferred to nitrocellulose membranes using the iBlot Dry Blotting System  at 23 volts for 7 minutes. The blots were blocked with StartingBlock T20 (Thermo Fisher Scientific Inc). SPCA1 (RS-1 #227) or SPCA2 (orange #2) antibodies were diluted 1/2000 in StartingBlock T20 and the blots were incubated over night at 4°C. After washing they were incubated 1 hr at RT with 1/50000 HRP goat anti-rabbit #31460 (Thermo Fisher Scientific Inc) and washed. Blots were developed using Pierce’s Supersignal  using the protocol provided by the manufacturer. Developed film was imaged and bands quantitated with a Gel Doc EZ imager .6 cells from cell culture were washed three times with sterile HBBS (Invitrogen) then immediately lysed in buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM Na3EDTA, 1 mM EGTA, 5 mM Na4P2O7, 1 mM Na3VO4, 10 mM NaF, pH 7.4 with 1% SDS in the presence of protease inhibitor cocktail (Roche). Protein concentrations were assayed using a bicinchoninic acid assay kit (Pierce). Approximately 50 µg of protein was loaded onto a NuPAGE gel for SDS-PAGE Analysis and Western blotting. Immunoblots were made on 0.45 µm nitrocellulose membrane (Bio-Rad). Antibodies for mouse SPCA2, Orai1 and GAPDH were incubated with the membrane overnight at 1∶1000 dilution in 1×PBST (PBS plus 0.2% Tween). Membranes were washed five times in PBST then mouse or rabbit secondary antibody conjugate to HRP was added  at 1∶2000 for one hour at room temperature on a rotator and the membranes were washed three times with PBST then four times with normal PBS to remove Tween residue. Blots were visualized using ECL kit (Pierce) on an Intelligent Dark Box Imager (Fuji Film).1×102 fragment of goat anti-mouse IgG(H+L), and Red anti-rabbit at 1∶1000 Alexa Fluor A11070 594 F(ab’)2 fragment of goat anti-rabbit IgG (H+L) . The slides were washed 3 times with blocking buffer and mounted with VectaShield (Vector Laboratories). Slides were viewed and photographed on Zeiss Axio Scope.A1 fluorescence microscope .Mammary tissue was fixed in Tellyesniczky’s fixative  for 5 h at room temperature Monolayer cultures were washed three times with 1×PBS prior to fixation in 4% paraformaldehyde and subsequent permeation in blocking buffer (1% BSA and 0.5% Triton-X in 1xPBS). Cells were then blocked and permeabilized in 0.1% Triton and 1% BSA in 1xPBS and washed three times in 1xPBS. Cells were incubated with antibodies to SPCA2 Mammospheres were fixed in ice-cold methanol and acetone  for five minutes on ice pLK0.1 plasmids were obtained from Sigma (Mission shRNA constructs) for SPCA2 and Orai1. HEK293T cells were inoculated with 1 µg plasmid DNA for each accessory packaging protein for lentiviral packaging. Media was removed from the cells after 72 hours and virus was purified using Lenti-X (Sigma) per the manufacturer’s instructions. Approximately 10 µg of virus was added to each well of a 6 well plate containing SCp2 cells at 50–70% confluence and allowed to incubate for 48 hours. The virus was then removed and cells were washed then inoculated with new media containing 500 µg/ml of puromycin for selection over 3 days. Once the cells were selected, they were grown in the selection media for two weeks then discarded. Each experiment was done within 5 passages of the cells to ensure complete RNA inhibition.2, 10 mM glucose at pH 7.4) containing 2 mM CaCl2. After incubation at room temperature for 20 minutes, cells were washed briefly in imaging buffer without calcium and 0.15% EGTA. Cells were then washed twice in imaging buffer without calcium to wash away residual EGTA and Ca2+. For mammosphere imaging, protocols were kept the same, however, after FURA2 loading, two 2 mM Ca2+ washes were added with slow agitation to remove FURA2 from the Matrigel, prior to washing in imaging buffer without calcium. Store dependent (SOCE) and independent (SICE) Ca2+ entry was measured as described 2+ to nominally Ca2+ free recording buffer. Thapsigargin (2 µm) or ionomycin (2 µm) was added where indicated, and followed by readdition of 2 mM Ca2+ to measure store-dependent Ca2+ influx. For SICE, fluorescence was recorded from cells placed in nominally Ca2+ free recording buffer, followed by addition 2 mM Ca2+, activating store-independent Ca2+ influx.Cells were cultured as a monolayers or mammospheres on 25 mm circular coverslips. Briefly, cells were washed in sterile HBBS (Invitrogen) for five minutes at a time, three times. After washing, FURA2-AM (Invitrogen) was added at a final concentration of 1 µg/ml in imaging buffer Click here for additional data file.Figure S2Morphology and Growth of SCp2 cells after transfection with shRNA constructs. A. Growth of SCp2 cells after transfection with lentivirus carrying empty vector or shRNA against SPCA2 or Orai1 was monitored using MTT assay as described in Methods. No significant differences were observed following knockdown. B. Morphology of SCp2 cells following knockdown of SPCA2 and Orai1 is similar to that of control cells.(TIF)Click here for additional data file.Figure S3Confocal sections of Immunofluorescence staining in Orai1 (A) and SPCA2  knock down SCp2 cells. A. Consecutive optical sections, starting from the bottom, of SCp2 cells knocked down for Orai channels stained with E-cadherin (red), SPCA2 (green) and DAPI (blue). Note the formation of tight junctions indicated by E-cadherin stain and normal shape of the cells. B. Consecutive optical sections, starting from the bottom, of SCp2 cells knocked down for SPCA2 stained with Orai1 (green) and DAPI (blue). Note the separation of Orai1 stain from the nucleus at the bottom and top sections. C. Confluent SCp2 cells treated with shEV (top) or shSPCA2 (bottom) stained for Orai1 (green) and DAPI (blue). Note the change in Orai1 localization from the cell boundaries (top) to circumnuclear (bottom).(TIF)Click here for additional data file.Figure S42+ in SCp2 cells.Estimation of Stored Ca A. SCp2 cells were transferred to nominally Ca2+ free medium at time 0 and thapsigargin was added between 5–30 seconds as indicated. Ca2+ release was monitored by Fura2 (340/380 ratio). B. SCp2 cells were transferred to nominally Ca2+ free medium or not, at time 0 as indicated. Baseline Ca2+ before addition of thapsigargin was unchanged (inset i) Thapsigargin was added and Ca2+ release was monitored as fold-change relative to starting 340/380 ratios. The difference between the traces, indicated by gray shading, was plotted in inset ii. This indicates maximal SOCE resulting from thapsigargin mediated store release. Note that it is smaller than Ca2+ influx observed in (TIF)Click here for additional data file.Movie S1Consecutive confocal sections of a mammosphere immunostained with antibody against E-cadherin (red) and SPCA2 (green), with DAPI stain of nuclei (blue).(GIF)Click here for additional data file.Movie S2Consecutive confocal sections of a mammosphere immunostained with antibody against Orai1 (red) and SPCA2 (green), with DAPI stain of nuclei (blue).(GIF)Click here for additional data file.Movie S3Consecutive confocal sections of a mammosphere immunostained with antibody against Orai1 (red) and SPCA2 (green), with DAPI stain of nuclei (blue).(GIF)Click here for additional data file.


Intracellular Ca2+ is crucial for the orderly cell cycle progression and plays a vital role in the regulation of cell proliferation. Recently, it was demonstrated by in vitro and in vivo studies that nucleoplasmic Ca2+ regulates cell growth. Even though the mechanism by which nuclear Ca2+ regulates cell proliferation is not completely understood, there are reports demonstrating that activation of tyrosine kinase receptors (RTKs) leads to translocation of RTKs to the nucleus to generate localized nuclear Ca2+ signaling which are believed to modulate cell proliferation. Moreover, nuclear Ca2+ regulates the expression of genes involved in cell growth. This review will describe the nuclear Ca2+ signaling machinery and its role in cell proliferation. Additionally, the potential role of nuclear Ca2+ as a target in cancer therapy will be discussed.Calcium (Ca The activation of such receptors recruits several second messengers, including phospholipase C (PLC) that, once activated, cleaves phosphatidylinositol 4,5-biphosphate (PIP2) producing diacylglycerol and inositol-1,4,5-trisphosphate (InsP3). InsP3 then binds to the InsP3 receptor (InsP3R), activating its channel to release Ca2+ from the endoplasmic reticulum. Once in the cytosol, Ca2+ can participate in several intracellular cascades and activate another class of Ca2+ channels, the ryanodine receptor (RyR), triggering a process denoted Ca2+-induced Ca2+ release [2+ in a wide range of mammalian cell types [2+ channels, the two pore channels (TPCs), are activated by nicotinic acid adenine dinucleotide phosphate (NAAD) which promotes release of Ca2+ from acidic organelles [2+ signaling in mammalian cells as well [2+ channels coordinate cellular responses mediated by Ca2+, both in health and disease. However little is known regarding the interaction of intracellular Ca2+ channels in the regulation of nuclear Ca2+ signaling.Intracellular calcium . The nuiewed in ), and thiewed in . With th2+ has biological effects that differ from those mediated by increases in cytosolic Ca2+[2+ is specifically increased in the nucleoplasm are a topic of debate. It was initially proposed that nuclear Ca2+ signaling would occur by passive diffusion of cytosolic Ca2+ across the nuclear envelope into the nucleoplasm. However, as it will be discussed in more detail, the nuclear interior has all the machinery required to produce localized Ca2+ signals, supporting the concept of the nuclear compartment as an independent apparatus to trigger Ca2+ signals. Moreover, the mechanisms and pathways by which localized Ca2+ signals in the nucleus regulate cell growth have only recently been investigated. The emerging model . Althou2+ rich compartment, accumulating Ca2+ via a Ca2+-ATPase pump (SERCA) and a Na+/Ca2+-exchanger [3[2+-ATPase pump was shown to be present only in the outer membrane of the nuclear envelop, while the Na+/Ca2+-exchanger, is expressed in the inner membrane [2+ channels, the RyRs appears to be present on both leaflets of the nuclear envelope [3Rs in the inner and outer membrane [Aplysia neurons, depolarization is the signal that triggers the translocation of CD38 to the nucleus [3, including PIP2, and PLC [The nuclear envelope itself is a Caxchanger -38 and rhanger [3,39, cADPhanger [3, and NAAhanger [3. The Ca2membrane . Regardienvelope . Similarmembrane ,44,45. Imembrane . In Aply nucleus . Moreove and PLC , and thi and PLC .2+ signaling machinery. The nucleoplasmic reticulum represents another specialized cellular compartment involved in regulation in time and space of specific intracellular Ca2+ signaling events. For instance, both the InsP3R and the RyR are found in the nucleoplasmic reticulum [3-Kinase (IP3KB), the isoform that inactivates the InsP3 by phosphorylating it, was also reported to be located in the nucleoplasmic reticulum, where it may function to terminate the InsP3 mediated Ca2+ signal [2+ signaling regulatory domain deep in the nucleus, along the nucleoplasmic reticulum, providing further spatial control of Ca2+ within this cellular compartment [However, the nuclear envelope is not the only nuclear site containing the Caeticulum ,23. Impo+ signal . In addi+ signal . Therefopartment ,51,52.2+ signals. Several in vitro studies have shown that InsP3 releases Ca2+ directly from the nuclear envelop into the nucleus [2+ release, via an InsP3-dependent mechanism [2+ release via InsP3R that activates nuclear CaMKII triggering HDAC5 phosphorylation and its nuclear export [3, cADPR can also increase Ca2+ in isolated cell nuclei [Corroborating these findings, there is a growing body of data demonstrating that the nucleus has the capacity to independently generate Ca nucleus ,44,53,54echanism . In cardr export . This sil nuclei ,39,40.3 generates nuclear Ca2+ signaling is via translocation of activated RTKs from the plasma membrane to the nuclear interior. For instance, it was shown that IGF-1 and integrins caused PIP2 breakdown in the nucleus but not at the plasma membrane [3 formation that was followed by nuclear Ca2+ signals [3 there [One of the proposed mechanism by which InsPmembrane . Similar signals ,27  [2+[2+ can negatively regulate the activity of transcription factors, such as TEAD [2+ has also been implicated in modulating cardiac hypertrophy [2+ was shown to bind to and directly regulate DNA structure [2+ signaling pathway came from studies showing that in skeletal muscle cell, two-photon photorelease of caged Ca2+ near the nucleoplasmic reticulum was found to elicit a Ca2+-induced Ca2+ release event within the nucleus [2+ signals specifically control the progression through early prophase, showing that nucleoplasmic Ca2+ regulates cell proliferation [Once in the nucleus, Cay of PKC  and CaMKy of PKC . Nuclearin (CBP) . Transcr(CBP) [2+. On the  as TEAD . Moreoveertrophy ,51 and wtructure . Another nucleus . More referation .2+ signals have an important role throughout the mammalian cell cycle and are especially important in early G1 and G1/S and G2/M transitions [2+ transient occurring just prior to entry into mitosis, and the second one occurring during the metaphase-anaphase transition [2+ is the most prominent messenger required through these cycle points [2+ have also been implicated in cell cycle progression as well [It has been long recognized that Cansitions , with thansition ,64. Indee points ,66 and d as well ,68.2+ binding protein parvalbumin has been used to study the role of Ca2+ signals in the regulation of the cell cycle. This protein is normally expressed in skeletal muscle and neurons [2+[2+ and study cellular growth, showed that reducing Ca2+ slowed progression through the cell cycle [2+ on proliferation correlate with the subcellular compartment where Ca2+ is released. Using parvalbumin variants, selectively targeted to distinct intracellular Ca2+ rich compartments, it was found that buffering mitochondrial Ca2+ inhibits apoptosis and accelerates hepatocyte proliferation [2+ was shown to retard liver regeneration and progression through the cell cycle after partial hepatectomy [2+ can increase through a number of mechanisms, it is believed that, in this compartment, Ca2+ may have different effects on cell growth [2+ is essential for liver cell line proliferation, and is necessary in particular for progression through early prophase [3 in the nucleus to initiate nuclear Ca2+ signals [2+ to particular sites within the nucleus.Heterologous expression of the Ca neurons , and is urons [2+. The firll cycle . Howeverferation . In contatectomy . Since cl growth ,73,74. Oprophase . It was prophase . Moreove signals ,27  as a novel target of nuclear Ca2+[2+ buffer protein parvalbumin targeted to the nucleus, from genes in cells expressing a mutated form of nuclear-targeted parvalbumin which has one of the two Ca2+-binding sites inactivated. The authors identified thirteen genes whose expression was affected by a small alteration in nuclear Ca2+ concentration. LGMN was one of such genes and upon further validation was demonstrated to be regulated by nuclear Ca2+ signals at the transcriptional level. LGMN was first recognized in plants [2+ was buffered in the nucleus of the cells, LGMN expression decreased, impairing cell proliferation [2+ signals regulate cell proliferation at least in part through the modulation of gene expression (Figure 2+ that are involved in cell proliferation still remain to be described.Although, the proteins that link nuclear Calear Ca2+. Using Rn plants  and laten plants . It is pn plants . For insn plants ,79. So, feration . Addition Figure . Other t2+ buffering could be used in conjunction with radiotherapy as a therapeutic potential for the treatment of carcinoma. Ionizing radiation concomitant with nuclear Ca2+ buffering showed superior outcome, compared to irradiation alone [2+ buffering in the proposed antitumor therapy was shown to be due to changes caused in expression level of genes involved in the regulation of cell proliferation [2+ reduced the rate of tumor cell proliferation, without affecting cells from normal tissue [2+ towards controlling cancer cell growth. Further studies are required to determine the mechanistic basis for the differential sensitivity of normal versus cancer cell proliferation to changes in nuclear Ca2+. Nonetheless, these findings suggest that buffering nuclear Ca2+ could be one strategy employed to inhibit the growth of tumors without affecting normal tissue, either alone or in association therapy.Altered nuclear morphology is a common feature of many cancers  and it hon alone . Corroboferation . Moreovel tissue , suggest2+ is important to several signaling pathways among virtually every cell type. The central mechanism by which Ca2+ regulates protein functions depends on how and where it is released into the cell. The role of nuclear Ca2+ in cell proliferation was demonstrated in vitro by showing that nuclear Ca2+ buffering reduced proliferation rate through blocking cell cycle in G2/M phase. It was also demonstrated that nuclear Ca2+ plays a role on tumor growth in vivo and it can alter the expression of genes involved in cell proliferation. Moreover, modulation of nuclear Ca2+ signaling was shown to be a potential target to treat cancer. However further studies are needed to better understand how nuclear Ca2+ can be generated and how it regulates cell proliferation and cell cycle progression. These findings would have strong potential as therapeutic targets in degenerative diseases or cancer.CaThe authors declare that they have no competing interests.All authors contributed in the conception and writing of the manuscript. All authors edited and approved the final version.


Hypertension and ageing are associated with altered Ca2+ handling including possible activation of Ca2+-dependent Cl− channels. Our study was aimed to determine Ca2+-dependent Cl− channels contribution to norepinephrine-induced contraction during hypertension and ageing. Norepinephrine-induced concentration-response curves of femoral arteries from 6- and 12-month-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were recorded using wire myograph. Pretreatment with Ca2+-dependent Cl- channel inhibitor indanyloxyacetic acid 94 [R(+)-IAA-94](IAA) attenuated norepinephrine-induced contraction in all groups, but relatively more in WKY than SHR arteries. The attenuation of norepinephrine-induced contraction after Ca2+-dependent Cl− channels blockade was partially reduced in 12-month-old WKY rats, but substantially diminished in 12-month-old SHR. IAA effect was enhanced after NO synthase inhibition but decreased by ageing. In 20-month-old WKY rats norepinephrine-induced contraction was not affected by IAA but was almost abolished after cyclooxygenase inhibition by indomethacin or niflumic acid. In conclusion, contribution of Ca2+-dependent Cl− channels to norepinephrine-induced contraction diminished with age, hypertension development, and/or NO synthesis inhibition. Ca2+-dependent Cl− channels are important for maintenance of normal vascular tone while their inactivation/closing might be a pathological mechanism.The activation of Ca The link between the opening of Ca2+ stores and L-VDCC could be mediated either through the elevation of intracellular Ca2+  [Ca2+]i levels or might involve other mechanisms such as opening of Cl− channels or closure of K+ channels [2+ influx through L-VDCC and is modulated by a negative feedback pathway involving activation of large-conductance Ca2+-dependent K+ channels. We demonstrated that these K+ channels play an important role in modulating vascular contraction in spontaneous hypertension [− in vascular cells is −26 mV [2+-dependent Cl− channels play a role in Ca2+ uptake to sarcoplasmic reticulum in smooth muscle because the administration of Cl− channel blockers NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) and IAA (indanyloxyacetic acid 94 (R(+)-IAA-94) almost completely blocked the Ca2+ uptake [− conductance is activated by the enhancement of either Ca2+ release from Ca2+ stores or Ca2+ influx [2+-dependent Cl− channels exist in two states, open and closed, with a relatively long mean open time. Some of the agents that inhibit Ca2+-dependent Cl− channels interact directly with the open channel, which suggests that the most likely role of Ca2+-dependent Cl− channels in smooth muscle is to produce membrane depolarization and contraction to neurotransmitters [2+ release from Ca2+ stores and Ca2+ influx should be altered by the blockade of Ca2+-dependent Cl− channels. It has been shown that Ca2+-dependent Cl− channels play a major role in the response to norepinephrine in the portal vein [− channels by Ca2+ release from intracellular stores might determine the maximal contraction by influencing the magnitude of membrane depolarization during agonist-induced contraction.The stimulation of vascular smooth muscle cells with norepinephrine leads to the release of Cachannels –3. Braydchannels  showed trtension . Calculas −26 mV . Further+ uptake . It is esmitters . Therefotal vein  or in thtal vein . Chloridtal vein . Pacaud tal vein  suggeste2+-dependent Cl− channels in aortic smooth muscle cells occurs after caffeine-induced Ca2+ release without Ca2+ influx, but the repetitive activation of Cl− channels depends on Ca2+ influx through L-VDCC. Moreover, the progressive disappearance of caffeine-induced Ca2+ release and Ca2+-dependent Cl− currents in smooth muscle cells treated with NO donors could result from NO-induced inhibition of ryanodine- and caffeine-sensitive Ca2+ channels and/or L-VDCC [− conductance are involved in the L-NAME-induced contraction and the blockade of Cl− channels abolished the L-NAME-induced contractions [− channel activation. Using small mesenteric arteries Matchkov et al. [− current which is dependent on Ca2+ and cGMP and that the NO is necessary for the activation of this current, which leads to vasomotion and can participate in smooth muscle cell synchronization. This Ca2+-dependent and cGMP-dependent Cl− current is sensitive to Zn2+ ions but less sensitive to conventional Cl− channel blockers.The activation of Car L-VDCC . This meractions . On the v et al.  and Boedv et al.  showed tβ-adrenoceptor stimulation [2+ entry and cell Ca2+ handling [2+ influx through L-VDCC in the control of vascular tone and blood pressure can be demonstrated both under the in vivo and in vitro conditions as we have previously reported in conscious SHR and their isolated femoral arteries [There are numerous reports on altered vascular function in hypertension and/or ageing in rats (for review see ). These mulation , 23, chahandling , 25, andarteries –28.2+-dependent Cl− channel-sensitive component of norepinephrine-induced contraction during the ageing and hypertension. Our hypothesis was that norepinephrine-stimulation of vascular smooth muscle opens/activates Ca2+-dependent Cl− channels in SHR vessels to a larger extent than in those of WKY. The NO synthase inhibition was used to augment the magnitude of norepinephrine-induced contraction as well as to modify the contribution of the above ion channels to this contraction. IAA was used to inhibit Ca2+-dependent Cl− channels in femoral arteries isolated from four groups of rats—adult WKY rats, aged WKY rats, adult SHR, and aged SHR. The second aim of our study was to compare the contribution of Ca2+-dependent Cl− channels and endothelium-derived constricting factor (EDCF) to norepinephrine-induced contraction (including their age-dependent changes). This was possible to examine only in femoral arteries because EDCF is produced in femoral but not in small mesenteric arteries [2+-dependent Cl− channels by IAA and after the inhibition of cyclooxygenase by indomethacin, which is the main enzyme producing endothelium-derived constricting factor (EDCF).Our aim was to quantify the changes in Caarteries . We invead libitum). All procedures and experimental protocols, which were approved by the Ethical Committee of the Institute of Physiology, Academy of Sciences of Czech Republic, conform to the European Convention on Animal Protection and Guidelines on Research Animal Use.Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were sacrificed at the age of 6 months (adult WKY rats and adult SHR) and 12 months (aged WKY rats and aged SHR rats). The second series of experiments was carried out in 20-month-old WKY rats. Animals were housed under standard laboratory conditions . The myograph chamber was filled with modified Krebs-Henseleit solution  and bubbled with 95% O2 and 5% CO2. The inner arterial diameter was set to be 90% of the diameter predicted for the pressure of 100 mm Hg in the wire myograph. During 30 min of stabilization the vessels were left to achieve their basal tone. To provide the maximal depolarization-induced contraction vessels were incubated with a depolarizing solution (modified Krebs-Henseleit solution with 124 mM K+ but without Na+).Animals were anesthetized with ether and blood pressure was measured directly by the puncture of carotid artery. The animals were killed by overdose of CO−8 to 10−4 mol/L) and then acetylcholine-induced relaxations (3.10−8 mol/L and 10−6 mol/L or 3.10−6 mol/L) of norepinephrine-precontracted vessels were measured. One segment with preserved endothelium was studied in each rat. After repeated washing and stabilization of basal tone norepinephrine-induced concentration-response curves were again determined in the same artery segment in the presence of NO synthase inhibitor Nω-nitro-L-arginine  or in the presence of Ca2+-dependent Cl− channel blocker indanyloxyacetic acid 94 (R(+)-IAA-94, IAA, 10−5 mol/L, preincubation time 10 min). Finally, these concentration-response curves were measured in the presence of both inhibitors L-NNA and IAA.The femoral arteries isolated from adult and aged WKY rats and SHR were studied in the first part of our experiments. Subsequent arterial contractions were induced by cumulative doses of norepinephrine (3.10−8 to 10−4 mol/L) followed by arterial relaxation elicited by acetylcholine application (10−8 mol/L and 10−6 mol/L) were first measured in the absence of inhibitors. Thereafter norepinephrine-induced concentration-response curves were determined in the presence of Ca2+-dependent Cl− channel blocker indanyloxyacetic acid 94 (R(+)-IAA-94, IAA, 10−5 mol/L, preincubation time 10 min) and after 30 min washing period again in the presence of cyclooxygenase inhibitor indomethacin . In another group of vessels norepinephrine-induced concentration-response curves were determined in the presence of niflumic acid  which was used as cyclooxygenase inhibitor and Ca2+-dependent Cl− channel blocker.In the second part of our study we used femoral arteries isolated from 20-month-old WKY rats. Arterial contraction induced by cumulative doses of norepinephrine , and slope—were calculated for each norepinephrine-induced concentration-response curve. Data are presented as mean ± S.E.M. Statistical analysis was performed with one-way ANOVA and post hoc least significant difference test.All chemicals were obtained from Sigma . R(+)-IAA-94 and niflumic acid were dissolved in ethanol maintaining the final concentration of ethanol in the chamber under 1%. Indomethacin was dissolved in 170 mM Nae values . PharmacBlood pressure was higher in SHR than WKY rats without any significant age-dependent increase. The inner diameter of studied femoral arteries was always significantly smaller in SHR than in WKY rats, but it increased with age in both rat strains .Emax) was enhanced in vessels from SHR compared to WKY vessels. Ageing increased the maximal contraction in vessels from SHR only (−8 mol/L relaxed the arteries of adult and aged WKY as well as those of adult SHR by about 50%. The vessels from aged SHR relaxed by 37% only (−6 mol/L in WKY and 3.10−6 mol/L in SHR) the maximal acetylcholine-induced relaxation of isolated femoral arteries was only slightly attenuated in SHR arteries  was enhanced and Emax was impaired by IAA pretreatment of WKY vessels compared to the control conditions. In contrast, there were no significant changes in norepinephrine sensitivity of concentration-response curves in SHR vessels  and reduced Emax more in WKY than in SHR vessels  responsible for the residual part of norepinephrine-induced arterial contraction seen mainly in SHR vessels remains to be determined. Resting membrane potential of vascular smooth muscle cells of SHR is increasing during the hypertension development and stays significantly higher compared to normotensive WKY rats [2+-dependent Cl− channels to a larger extent in vascular smooth muscle of SHR.We have investigated the effect of ageing and hypertension on the participation of Cale cells . HoweverWKY rats . Contrar2+-dependent Cl− channel inhibitor, but it has also a relatively high affinity to L-VDCC [2+-dependent Cl− channels blockers also activate large-conductance Ca2+-activated K+ channels [2+-activated K+ channels . To our+ concentration could augment cyclooxygenase 2 expression as it was demonstrated in cells with increased Na+i/K+i ratio [We have recently demonstrated  that a ci ratio . Neverthi ratio . Thus th2+-dependent Cl− channels are involved in a major part of norepinephrine-induced contraction of adult and aged WKY arteries, but the contribution of these channels to norepinephrine-induced contraction is decreasing with age and/or hypertension development as was shown using the arteries of 20-month-old WKY rats in which the blockade of Ca2+-dependent Cl− channels was without effect on norepinephrine-induced contraction . It remains to be determined which other cyclooxygenase-sensitive mechanism is responsible for the progressive age-dependent augmentation of norepinephrine-induced contraction of large arteries in ageing and hypertension.Catraction . This is2+-dependent Cl− channel inhibition, the inhibition of cyclooxygenase with indomethacin led to the attenuation of norepinephrine-induced contraction of femoral arteries of 20-month-old WKY rats. In parallel experiments with vessels of these old rats, the application of niflumic acid led to the attenuation of norepinephrine-induced contraction as well. Niflumic acid is considered to be a Ca2+-dependent Cl− channel inhibitor as well as cyclooxygenase inhibitor. Our experiments clearly indicated that niflumic acid is a potent cyclooxygenase inhibitor and that the results obtained with niflumic acid in other studies should be considered carefully. Unfortunately, we were not able to investigate the effect of niflumic acid after the administration of indomethacin on norepinephrine-induced contraction because indomethacin alone almost abolished the contraction to norepinephrine.In contrast to the lack of effects of Ca2+-dependent Cl− channel-sensitive component of norepinephrine-induced contraction. It is possible that the endothelial factors (such as NO) might contribute to the reduction of IAA-sensitive component of norepinephrine-induced contraction. Further studies with more specific compounds are needed to separate the Cl− current dependent on NO as shown by Matchkov et al. [2+-dependent Cl− current in vascular smooth muscle cells.The inhibition of NO synthesis pronounced the age- and hypertension-dependent reduction of Cav et al.  and Boedv et al.  from the2+-dependent Cl− channels was suggested as a possible therapeutic target in hypertension [2+-dependent Cl− channels is important for the maintenance of normal physiological state during norepinephrine-induced contraction, at least in femoral arteries. Further attention should be paid to the interactions of Cl− channels with other ion transporter systems. Recent data indicate that Cl− channels in vascular smooth muscle cells are under the control of inwardly directed Na+, K+, and 2Cl− cotransport (NKCC1). Numerous research groups demonstrated that the inhibitors of this carrier such as bumetanide and furosemide sharply diminished contraction of blood vessels elicited by norepinephrine or other vasoconstrictors . Increapromoter . It is iWKY rats . It woul2+-dependent Cl− channels to norepinephrine-induced arterial contraction, which diminishes with age, hypertension development, and/or inhibition of NO synthesis. Further studies are needed to provide the evidence for our hypothesis that Ca2+-dependent Cl− channels are important for the maintenance of normal physiological state in the vascular system. The loss or inactivation of these channels during ageing and/or hypertension development could be seen as a pathological mechanism.In conclusion, our study demonstrated a considerable contribution of Ca2+-dependent Cl− channels in norepinephrine-induced arterial contraction should be investigated in young normotensive and genetically hypertensive rats to reveal their potential role in the development of hypertension. The contribution of these ion channels to arterial contraction elicited by other vasoconstrictors (such as angiotensin II or endothelin-1) should also be studied. It would be desirable to evaluate the above mechanism in other forms of experimental hypertension.The contribution of Ca


S. cerevisiae  encodes a highly conserved eukaryotic protein of the mitochondrial inner membrane which is essential to control mitochondrial volume homeostasis. Indeed, LETM1 RNA interference causes severe mitochondrial changes that include massive matrix swelling, loss of cristae structure and network fragmentation in +-H+ exchanger (KHE)  causes an increase of mitochondrial volume that can be reverted by nigericin  and (ii) can be phenocopied by the addition of valinomycin   was inhibited by ruthenium red (RR)  while an antiporter with a stoichiometry of 2H+/Ca2+ can only catalyze Ca2+ efflux in energized mitochondria  but not with nigericin which catalyzes strictly electroneutral K+/Rb+-H+ exchange, and would therefore require a pH gradient to catalyze 86Rb+ accumulation. Thus, we think that the study of Tsai et al. has not provided an answer as to whether LETM1 mediates KHE in a reconstituted system.The study of Tsai et al. also addressed the question of whether K+ uptake; but how could mitochondrial dysfunction and swelling be caused by inactivation of a 2H+-Ca2+ exchanger mediating Ca2+ efflux? Mitochondrial Ca2+ distribution is governed by the balance of electrophoretic Ca2+ uptake through the MCU , a high-conductance channel which forms from dimers of the F-ATP synthase (Giorgio et al., +-Ca2+ exchange. Thus, at present there is a solid mechanism to explain mitochondrial dysfunction following LETM1 inactivation only if LETM1 takes part in KHE.There is no question that LETM1 inactivation causes severe mitochondrial dysfunction with depolarization and matrix swelling (Nowikovsky et al., 2+ uptake (Carafoli and Lehninger, 2+ release mechanism which can only lead to matrix Ca2+ depletion. Since yeast mitochondria undergo in situ swelling when LETM1 is inactivated -an event that cannot be ascribed to Ca2+ overload- we still favor the hypothesis that LETM1 catalyzes KHE in situ, as overwhelming evidence suggests (Nowikovsky et al., in situ is affected by protein interactions that are lost in the reconstituted system, and thus that LETM1 catalyzes RR-insensitive, electroneutral exchange of H+ with both K+ and Ca2+, an issue that should be addressed in future studies of this fascinating problem.The LETM1 gene has been evolutionarily conserved from yeast to trypanosomes, to Drosophila and mammals (Nowikovsky et al.,


The model reproduced typical manifestations of this leukaemia, including presence of extramedullary masses and central nervous system involvement, and the original phenotype, karyotype and genotype of leukaemic cells were retained in vivo. Recently Polo-Like Kinase 1 (PLK1) has emerged as a new candidate drug target in AML. We therefore tested our PLK1 inhibitor NMS-P937 in this model either in the engraftment or in the established disease settings. Both schedules showed good efficacy compared to standard therapies, with a significant increase in median survival time (MST) expecially in the established disease setting . Importantly, we could also demonstrate that NMS-P937 induced specific biomarker modulation in extramedullary tissues. This new in vivo model of CD56+ AML that recapitulates the human tumour lends support for the therapeutic use of PLK1 inhibitors in AML.CD56 is expressed in 15–20% of acute myeloid leukaemias (AML) and is associated with extramedullary diffusion, multidrug resistance and poor prognosis. We describe the establishment and characterisation of a novel disseminated model of AML (AML-NS8), generated by injection into mice of leukaemic blasts freshly isolated from a patient with an aggressive CD56 FLT3, CEBPA, NPM1, DNMT3, to cite only the most frequent events AML is a heterogeneous disease that accounts for about 30% of all adult leukaemias. The heterogeneity of AML was first evidenced by morphological and immunophenotypic analyses of the neoplastic cells. More recently an extensive genetic heterogeneity has also been identified, with a wide range of genetic alterations detected, such as chromosomal imbalances (42% of patients), recurrent chromosomal translocation/inversions/deletions involving different chromosomes (15%) and point mutations of specific genes, including CD56 (NCAM) expression is one of the prognostic markers in AML. It is heterogeneously expressed in different haematopoietic neoplasms, including AML, lymphomas and myelomas in vitro and in vivoConventional treatment for AML patients is based on a combination of anthracyclines and cytosine arabinoside (ara-C). However the heterogeneity of AML leads to response rates varying from 30% to 90%, evidencing the need for new treatment options, especially for poor prognosis AML subtypes + leukaemic cells (AML-NS8) obtained from an AML-M5a patient bearing trisomy 8 and 6. The tumour is aggressive in vivo in SCID mice, and in large part recapitulates the human disease with both medullary and extramedullary leukaemic infiltration. Furthermore the AML-NS8 cells responded favourably to our Polo-Like Kinase 1 (PLK1) inhibitor NMS-P937 compared to standard therapy, both in vitro and in vivo. These data suggest that PLK1 is a promising drugable target for AML therapy.New orthotopic xenograft models using leukaemic cells directly derived from patients have been developed and are an important tool for the preclinical evaluation of new drugs since they are likely to better represent the human disease −, HLA-DR+, CD33+, CD56+, CD7−, CD19−, α-napthyl acetate esterase (ANAE) positive monoblast like cells. Cytogenetic analysis (Q-banding) of the BM showed presence of 2 AML clones, respectively with trisomy 8 e double trisomy 8 and 6 . PCR analysis of BM sample showed negativity for AML1-ETO and CBFβ/MYH11 rearrangements, MLL partial tandem duplication, NPM1, CEBPA and FLT3-ITD and TDK mutations. The peripheral blood count was above 200000 wbc/µl. He was diagnosed with AML M5a according to FAB classification. The patient was enrolled in the NILG AML 01/00 clinical protocol “Risk-oriented therapeutic strategy for adult acute myelogenous leukaemia” . Leukapheresis was performed and a first dose of cytarabine infused to reduce the tumour burden. He died few weeks after appearance of first symptoms. Leukaemic cells were obtained from the leukapheresis after written informed consent and approval by the local institutional ethics committee . Mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation and aliquots frozen in 10% dimethyl sulphoxide.The adult patient AML-NS8 presented in hospital with a hypercellular bone marrow (BM) infiltrated by >90% with CD34NMS-P937 is a previously described PLK1 inhibitor 6 thawed AML-NS8 cells were injected intraperitoneally (ip) into irradiated NOD/SCID mice , as previously described 6cells/mouse. After 5 in vivo passages, the phenotype and genotype of collected and pooled cells were verified by flow cytometry, cytogenetic and SNP arrays analysis. These cells were frozen in aliquots for further studies.All procedures adopted for housing and handling of animals were in strict compliance with European and Italian Guidelines for Laboratory Animal Welfare. The protocol was approved by the Ethics of Animal Experiments Committee of Nerviano Medical Sciences. All efforts were made to minimize suffering. 50–100×106 pooled in vivo expanded AML-NS8 cells (from 5th passage) were transplanted into 5 weeks old SCID mice by tail-vein injection (iv). Animals were monitored for the insurgence of leukaemic signs, sacrificed when moribund and autopsied. For histopathology column, femour, sternum, whole skull, spleen, stomach, gut, liver, kidney, skin and lung tissues were collected. For flow cytometry, blood was taken by retro-orbital bleeds, BM was obtained by flushing it from femurs with PBS and spleen was mechanically dissected.Unless otherwise indicated 5×10in vivo experiments, in the engraftment setting (preemptive protocol) treatments started on day 4 after AML-NS8 iv injection. Mice were randomly assigned to one of the following groups: vehicle ip, cytarabine (75 mg/kg ip per day over 5 days for 4 cycles with 7 day rest), doxorubicin (3 mg/kg iv every 7 days for 3 cycles) or NMS-P937 (120 mg/kg os per day over 2 days for 4 cycles with a 10 day rest). Furthermore, in the established disease setting (therapeutic protocol), treatments started on day 20 after AML-NS8 injection when leukaemic dissemination was evaluable and mice were randomly divided into the following groups: vehicle ip, cytarabine , or NMS-P937 . Animals were monitored for clinical signs of disease and time of death recorded. All animals were autopsied.For Organs collected were fixed in 10% buffered formalin for 24 hours and 5% formic acid was added to decalcify bone structures. Tissues were prepared, stained with haematoxylin and eosin (H&E) and processed for immunohistochemistry as previously described in vivo expanded AML-NS8 cells from passage 5 were cultured at 0.5–1×106/ml in RPMI-1640 medium , supplemented with 20% foetal calf serum , 1 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and 10 ng/ml interleukin-3 (rhIL-3). To calculate doubling time, cells were seeded at 50×103cells/ml in duplicates in complete medium and counted daily in a Coulter Counter  for 8 days.Pooled 2. After 24 hours increasing concentrations of doxorubicin , cytarabine or NMS-P937  were added and incubation continued for further 72 hours. Cells were then processed by CellTiter-Glo assay  following the manufacturer’s instructions. Inhibitory activity was evaluated using the Symyx Assay Explorer  program. The concentration resulting in 50% inhibition (IC50) was calculated using the sigmoidal interpolation curve.AML-NS8 cells (2000 cells/well) were seeded in 384 well-plates in complete medium and incubated at 37°C and 5% COin vitro mechanism of action studies, AML-NS8 cells were seeded at 1.5×106/ml and treated with 0.2 µM NMS-P937 for 24 hours or 0.75 ng/ml Nocodazole for 16 hours. One part of cells were then lysed for western blotting and the other fixed in ethanol 70% and stained with Alexafluor 488 conjugated anti-phospho-Histone H3 antibody  and propidium iodide  for FACS analysis.For For standard immunophenotyping, cells in suspension were stained with CD3-PE, CD19-FITC, CD33-PE, HLA-DR-PE, CD10-FITC, CD123-PE, CD25-PE, CD11b-PE, CD18-FITC, CD56-PE , CD52-PE , CD117-PE , and analysed by FACSCalibur .Cells extracts and immunoblotting were performed as previously described in vivo (passage 5) and in vitro expanded AML-NS8 cells (passage 15) were genotyped using Affymetrix® Cytogenetics Whole Genome 2.7 M Array , as previously described Diagnostic, + AML for in vivo inoculation in mice. In this study AML-NS8 cells were directly derived from a patient with an aggressive CD56+ AML (whose characteristics are described in Since expression of CD56 (NCAM) has been observed in a significant proportion of cases of AML, ALL, MM and B-NHL and is associated with extramedullary involvement and poor prognosis, we have selected one case of aggressive CD56in vitro growing cell line for preliminary drug testing and mechanistic studies, AML-NS8 cells were put in culture in the presence of growth factors. In these conditions cells were able to expand in vitro, with a doubling time of 31 hours  mutations, MLL partial tandem duplication, AML1/ETO and CBFβ/MYH11 translocations] were detected in the diagnostic, in vivo and in vitro expanded cells by PCR analysis (data not shown).The karyotype of the al blood . These dal blood . The dous sample  and thiss sample . The in identify . No othein vivo and in vitro expanded cells. The genomic copy number (CN) profile obtained by SNP arrays on the diagnostic sample showed a number of deletions on chromosome loci 3q26, 9p21, 10p12, 11p14, 11q23 and 17p13, as well as a gain at the 14q21 locus, as detailed in in vivo expanded cells revealed that they were genetically stable with respect to the diagnostic leukapheresis sample, since they showed identical number and sizes of deletions and gains of genetic material  and MTAP (methylthioadenosine phosphorylase). This biallelic loss was present on all samples  were tested. As shown in 6 cells was chosen for further experiments of disseminated leukaemia, since this schedule gave a reproducible MST of 29 days ±1 .In order to generate a model of disseminated leukaemia, AML-NS8 cells obtained from intraperitoneal passage 5 were inoculated by iv route in SCID mice. Two different AML-NS8 cell concentrations  of cytarabine is comparable in the two different schedules , NMS-P937 showed a better %T/C in the established disease (221% versus 175% in the engraftment setting). Histological analysis indicated that the PLK1 inhibitor significantly decreased (in 5 out of 7 cases) or abolished (in 2 out of 7 cases) leukaemic infiltration in the meninges and soft tissues compared to cytarabine or vehicle in a therapeutic protocol. These data were confirmed by anti-human HLA A,B,C immunohistochemical staining of the tissues .in vitro. As shown in in vitro and revealed also the increased level of phospho-NPM1 expression compared to untreated cells , a direct target of PLK1 in solid tumours ed cells . We next animals  and inte animals . These d animals .in vivo model of a primary disseminated AML, named AML-NS8, derived from the leukapheresis sample of a patient with a very aggressive CD56+ acute monoblastic leukaemia (M5a). In mice the cells engrafted well and expanded within the abdominal cavity with the production of both ascites and solid tumour masses. Expanded cells were very similar to the primary tumour in terms of morphology, immunophenotype and karyotype. Indeed Q-banding revealed that all leukaemic cells at the 5th passage ip carried the double trisomy 8 and 6 already present in the leukapheresis sample at diagnosis. Besides, no other cytogenetic abnormalities or mutations were detected and the more refined genotype study by copy number analysis in SNP arrays demonstrated their genomic stability. In contrast, the same cells expanded in vitro for 5–6 weeks showed several additional chromosomal focal deletions and gains. These data support the view that, whereas the in vitro establishment of cell lines induces multiple genetic alterations that accumulate with time, in vivo expansion does not generally do so with such high frequency in vivo passages suggests that these cells can indeed provide a predictive and reliable animal model of an aggressive AML.Acute myeloid leukaemia is a heterogeneous disease, classifiable in different subtypes, which presents with highly variable and sometimes multiple cytogenetic, molecular and phenotypic abnormalities. Some of these aberrations correlate with response to therapy and survival. Despite significant progress in the treatment of AML, relapse and resistance are still common, calling for more effective therapies, whose development would require animal models recapitulating the patho-physiological features of these different disease entities INK4 gene was detected already at diagnosis in AML-NS8 cells. Biallelic mutations or deletions of this tumour suppressor gene is a common event in cancer Interestingly a biallelic loss of the p16in vivo stabilisation, expanded AML-NS8 cells were injected iv to induce disseminated disease which led to animal death within 28–30 days. The tumour dissemination was analysed in all haematopoietic and non haematopoietic organs using a variety of techniques including immunohistochemistry, flow cytometry and MRI. The data showed a clear infiltration of neoplastic cells in BM, lung, soft tissues and meninges in about 90% of mice and presence of solid masses in 25% of cases. Besides, small infiltrates of neoplastic cells were also present in spleen, blood, kidney, and liver. The infiltration of AML-NS8 cells within meninges of both column and skull and the documented presence of tumoural masses always associated with bones suggested that leukaemic cells in BM induced bone resorption, with consequent invasion of meninges on one side and muscles on the other, forming solid neoplasms.After Interestingly the overall distribution and localisation of neoplastic cells in extramedullary compartment and formation of solid masses as well as CNS and periodontal space involvement are common characteristics observed in patients AML-M5 in vitro at nanomolar doses leading to G2/M arrest and increase of mitotic markers. A comparable activity was previously observed in several other AML cell lines, irrespective of FAB subtype in vivo disseminated AML-NS8 model, this novel oral targeted drug significantly prolonged survival compared to conventional therapies not only in the preemptive protocol, in which the compound was administered concomitantly with leukemic cells injection, but especially in the therapeutic protocol where the treatment started 20 days after tumour engraftment. With this schedule, better resembling the clinical condition of patients in treatment, our PLK1 inhibitor NMS-P937 had a stronger effect (MST = 62 days) compared to cytarabine (MST = 36 days) and vehicle (MST = 28 days) and produced a better %T/C than in the preemptive protocol (221% vs 175% respectively). Importantly in the therapeutic protocol, our PLK1 inhibitor significantly decreased or abolished leukaemic infiltration in meninges and soft tissues compared to cytarabine or vehicle. These promising results, even if obtained only in one case of aggressive AML, togheter with the efficacy of this inhibitor in other AML models AML-NS8 cells did not show genetic alterations therapeutically targetable so an alternative strategy could be represented by cell cycle inhibitors. PLK are a family of ser/thr kinases composed of 5 members, most of which are involved in cell cycle control. PLK1, that is the best studied of the family, phosphorylates cyclin B1, activates the cyclin B/Cdk1 complex and cooperates with Aurora kinases, thus controlling mitosis. It is also important for centrosome maturation, cohesion of sister chromatids and exit from mitosis in vivo mechanism of action of the drug and it indicates that the AML-NS8 model is manageable for mechanistic studies. Finally, we could also show the possibility of following extramedullary infiltrations by MRI and this represents an additional tool for studying drug efficacy.An important aspect in the evaluation of targeted therapies is the availability of reliable biomarkers to correlate the efficacy of a new drug to its mechanism of action and afterwards to translate in clinical practice. So, as suggested by Berg et al. + AML provides a relevant system for integrating drug screening with biomarker evaluation. The efficacy of our PLK1 inhibitor NMS-P937 in this model supports its clinical development in leukaemias.In summary, we demonstrate that this new mouse model of CD56Figure S1Cytogenetic analysis of AML-NS8 samples. (A) Bone marrow sample from patient at diagnosis. Cytogenetic analysis (Q-banding) showed the presence of 2 clones, one with trisomy 8 in 7 out of 22 metaphases (A1) and the other the double trisomy of chromosome 6 and 8 in 15 out of 22 metaphases (A2). (B) AML-NS8 cells expanded in mice. Cytogenetic analysis showed the double trisomy of chromosome 6 and 8. (C) FISH analysis. In vivo expanded cells cytocentrifuged and analyzed by FISH, confirmed the same evidence observed in B. The centromeres of chromosome 6 and chromosome 8 are stained in red and green respectively.(TIF)Click here for additional data file.Figure S2Histopathological analysis and Magnetic Resonance Imaging (MRI) of bone structures. SCID mice were inoculated iv with 5×106 AML-NS8 cells and sacrificed upon manifestations of terminal disease. Column and skull were collected and fixed for histological analysis by H&E staining. The skull was also visualised by magnetic resonance imaging. (A) column transversal section (H&E) at ×25 magnification. Massive neoplastic cells infiltration of the muscle (ms), epidural space/meninges (es/m) and vertebral bone marrow (BM) was evident accompanied by areas of bone resorption (arrows). The neoplastic cells are stained blue. Black bar, 1 mm. (B) skull transversal section (H&E) at ×10 magnification. Deep neoplastic infiltration of epidural space/meninges (es/m). Arrow indicates macroscopic mass growing on the skull surface. Black bar, 1 mm. (C) T2-weighted MR image of the skull transversal section. A large area of meningeal neoplastic infiltration (indicated in yellow) is visible and surrounds the whole brain.(TIF)Click here for additional data file.Figure S3Biomarker expression in meninges of vehicle or NMS-P937 treated animals. Skull from vehicle or NMS-P937 treated animals were collected, fixed and paraffin embedded. Serial sections were stained with H&E or antibodies against phospho-TCTP (pTCTP), phospho-Histone H3 (pH3) or phospho-NPM1 (pNPM1). Severe infiltration by leukaemic cells was evident in the epidural space and meninges (es/m) under skull bone (b). As already seen in tumor masses, and as aspected due to its mechanism of action, NMS-P937 abolished the expression of its direct substrate pTCTP and increased mitotic markers also in meninges. Representative pictures at ×100 are reported. Inserts show high magnification at ×400. Black/white bar, 200 µm.(TIF)Click here for additional data file.


ANXUR receptor-like kinases facilitate this growth by linking the intracellular growth machinery of pollen tubes to the status of the extracellular matrix via H Arabidopsis. Here, we show that over-expression of the ANXUR RLKs inhibits growth by over-activating exocytosis and the over-accumulation of secreted cell wall material. Moreover, the characterization of mutations in two partially redundant pollen-expressed NADPH oxidases coupled with genetic interaction studies demonstrate that the ANXUR RLKs function upstream of these NADPH oxidases. Using the H2O2-sensitive HyPer and the Ca2+-sensitive YC3.60 sensors in NADPH oxidase-deficient mutants, we reveal that NADPH oxidases generate tip-localized, pulsating H2O2 production that functions, possibly through Ca2+ channel activation, to maintain a steady tip-focused Ca2+ gradient during growth. Our findings support a model where ECM-sensing receptors regulate reactive oxygen species production, Ca2+ homeostasis, and exocytosis to coordinate ECM-performance with the internal growth machinery.It has become increasingly apparent that the extracellular matrix (ECM), which in plants corresponds to the cell wall, can influence intracellular activities in ways that go far beyond their supposedly passive mechanical support. In plants, growing cells use mechanisms sensing cell wall integrity to coordinate cell wall performance with the internal growth machinery to avoid growth cessation or loss of integrity. How this coordination precisely works is unknown. Previously, we reported that in the tip-growing pollen tube the ANXUR receptor-like kinases (RLKs) of the CrRLK1L subfamily are essential to sustain growth without loss of cell wall integrity in  2+ gradient. These tip-growing cells tightly coordinate the loosening and pressure-driven deformation of their extracellular matrix (ECM)—the cell wall in plant cells—by locally adding new membrane and cell wall materials. In pollen tubes, which grow at amazing speeds to effect fertilization in plants, a class of kinases called the ANXUR receptor-like kinases (RLKs) sense perturbations in cell wall integrity, and their loss leads to pollen tube rupture. Here, we gain new insights into the mechanism of cell wall surveillance by these RLKs in the model plant Arabidopsis. We show that over-expressing ANXUR RLKs over-activates exocytosis, causing an over-accumulation of secreted cell wall material that eventually leads to growth arrest. Moreover, we find that the ANXUR RLKs function upstream of NADPH oxidases, which are membrane-anchored enzymes that produce reactive oxygen species (ROS). Using H2O2- and Ca2+-sensitive reporters, we show that NADPH oxidases generate tip-localized H2O2 production, which is required to maintain a steady, tip-focused Ca2+ gradient that is essential for pollen tube growth. We postulate that ECM-sensing receptors, such as the ANXUR RLKs, regulate ROS production, Ca2+ homeostasis, and exocytosis to coordinate the status of the ECM with the cell's internal growth machinery.Tip-growing cells, such as plant root hairs and pollen tubes or fungal hyphae, are characterized by a tip-focused Ca In vivo the ECM contributes to the bulk, shape, and strength of many tissues and, therefore, plays a central role in development It is well established that growing animal cells control the biogenesis, deposition, and remodeling of their extracellular matrix (ECM). Arabidopsis receptor-like kinases (RLKs) of the Catharanthus roseus RLK1-like subfamily (CrRLK1L) have received increasing attention as putative sensors that coordinate cellular growth and CW integrity (reviewed in phox), are also called “respiratory burst oxidase homologues (Rboh)”, has been firmly established in various fundamental processes. These include localized lignin deposition 2 to produce superoxide radicals in the apoplast , disruption of FER leads to an impairment of ROS production and defective root hairs that burst ANXUR1 (ANX1) and ANX2, the two closest homologues of FER, triggers the rupture of pollen tubes (PTs), the tip-growing male gametophytes of flowering plants, resulting in male sterility fer root hairs, anx1 anx2 double mutant pollen form bulges and burst, failing to maintain their integrity during growth. This indicates that the FER and ANX RLKs could be cell-surface receptors that control CW integrity in tip-growing cells. In PTs, genetic evidence for the involvement of NADPH oxidases is lacking, but several studies have revealed a role for ROS during PT growth that remains to be precisely characterized. For example, it has been shown that either the use of ROS scavengers or the NADPH oxidase inhibitor diphenylene iodonium (DPI), or the down-regulation of a NADPH oxidase, reduces PT growth in tobacco Connections between members of the CrRLK1L and NADPH oxidase families have been proposed or established for THE1 and FER, respectively. For example, 2+ channels and for the generation of a tip-focused gradient of cytosolic free calcium [Ca2+]cyt, which is essential for polar growth rbohC root hairs still display a tip-focused Ca2+ gradient, showing that RbohC was not essential for its establishment 2+ signaling and the growth-inhibiting effect behind the tip by rigidifying the CW—are not mutually exclusive as they could recruit different forms of ROS at different times and in different locations.Because of the difficulty to image the dynamics of ROS production with good spatial and temporal resolution, and because of its multi-faceted impacts on CW properties and the activation of intracellular signaling, it is unknown how NADPH oxidase-dependent ROS control polar growth RbohH and RbohJ, demonstrate that the ANX RLKs function upstream of these NADPH oxidases. Furthermore, analyses of the genetically encoded H2O2-sensitive HyPer and Ca2+-sensitive YC3.60 sensors in NADPH oxidase-deficient pollen revealed that NADPH oxidases generate tip-localized, pulsating ROS that are responsible—possibly through activation of Ca2+ channels—for maintaining a steady, tip-focused Ca2+ gradient.In this study we show that over-expression of the ANX RLKs inhibits PT growth by the over-activation of exocytosis and the over-accumulation of secreted membrane and CW materials. Genetic interaction studies coupled with a phenotypic characterization of loss-of-function mutants of two partially redundant, pollen-expressed NADPH oxidases, Arabidopsis lines in vitro pollen germination and growth assays of homozygous lines that carry a single insertion of the constructs in the T3 generation revealed that ANX1-YFP and, to a lesser extent, ANX2-YFP inhibit pollen germination and PT growth compared to the wild type (WT)  and ANX2-YFP protein fusions are polarly localized in the PM at the tip of growing PTs in independent T1 transgenic ype (WT) , and S1.m the WT . Thus, bin vitro phenotypes impact the fitness of PTs in vivo, the male transmission efficiency (TEM) was assayed for each of the ANX-OX lines. Male transmission of ANX-YFP fusions was significantly decreased for all but one ANX2-OX line, showing that PTs over-expressing ANX-YFP fusion proteins are not as competitive as untransformed WT PTs . The remaining ANX1-OX PTs  had ceased to elongate and displayed PM invaginations at the PT tip as observed with both YFP and the lipophilic dye FM4-64 . PM invaginations were accompanied by thick extracellular deposits of CW material .Since the surface of both the PM and the secreted CW material increase at the tip, we hypothesize that the balance between endocytosis and exocytosis rates might be tilted towards exocytosis at the tip of ANX-OX PTs. This could be achieved by a decrease or increase in the rate of endocytosis or exocytosis, respectively, or by a combination of both. For example, CW accumulation and PM invaginations have also been reported for tobacco PTs that over-express the phosphatidylinositol-4-phosphate 5-kinases PIP5K4, PIP5K5, and PIP5K6 10sec) reached on average 47%±9% of the maximum relative fluorescence with a PT growth rate of 4.02±1.41 µm min−1  treatment, a well-known inhibitor of exocytosis, ANX1-YFP is inserted at the apical PM via exocytosis . Thus, w n = 18; . No corr10sec was significantly higher than in the complemented line , while their PT growth rate was significantly decreased to 1.34±0.68 µm min−1 and 1.78±0.53 µm min−1, respectively  and RbohJ (At3g45810), sharing 81% amino acid identity, define a subgroup that is preferentially expressed in pollen (rbohH-1 (GABI_028G04), rbohH-3 (SALK_136917), rbohJ-2 (SAIL_31_D07), and rbohJ-3 (SALK_050665), which show little or no expression of the corresponding gene (rbohJ-2 and rbohJ-3 mutant plants behaved like WT (∼8.5% bursting), while around 57% of PTs of single rbohH-1 and rbohH-3 mutant plants ruptured in vitro  was crossed to rbohH-1 rbohJ-2 double mutant plants. Partially male sterile plants homozygous for rbohH-1 rbohJ-2 and homozygous for ANX1-YFP were retrieved in the F2 generation. Intriguingly, rbohH-1 rbohJ-2 pollen strongly expressing ANX1-YFP behaved exactly like rbohH-1 rbohJ-2 pollen with germination and PT rupture rates of ∼70% and 82%, respectively (n>100) of rbohH-1 rbohJ-2 plants homozygous for ANX1-YFP displayed CW accumulation or PM invagination, phenotypes observed in ANX1-OX PTs. To independently confirm these results, we directly transformed rbohH-1 rbohJ-2 mutant with ANX1-YFP fusion. Four independent, partially male sterile rbohH-1 rbohJ-2 transgenic lines homozygous for ANX1-YFP were recovered in the T2 generation. Again, neither CW accumulation nor PM invagination was observed in growing PTs (n>100 PTs for each), which eventually ruptured similar to the rbohH-1 rbohJ-2 PTs without the ANX1-OX construct. Furthermore, FRAP analyses also showed that the fast recovery rate at the apical PM observed in ANX1-OX PTs was suppressed in the rbohH rbohJ background as I10sec for rbohH rbohJ PTs over-expressing ANX1-YFP was similar to the complemented line chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) to stain PTs of WT and rbohH rbohJ double mutants. Fluorescence quantification of the apical cytoplasm in growing PTs treated for 5 minutes with 2 µM CM-H2DCFDA showed that PTs of the rbohH-1 rbohJ-2 and rbohH-3 rbohJ-3 double mutants displayed only 25% of the CM-H2DCFDA-derived fluorescence signal observed in WT PTs  , the HyPer activity measured as the ratio of F488/F405 was stronger in the shank of PTs than at the tip  . These r the tip . We hypotant PTs , the str2O2 production observed at the tip-periphery, we transformed partially sterile rbohH-3 rbohJ-3 plants with a green fluorescent protein (GFP)-RbohH fusion. Forty-four independent T1 transgenic lines out of 50 displayed rescue of sterility with WT-like elongated siliques . In vitro pollen growth assays confirmed that the rbohH rbohJ bursting phenotypes were rescued by GFP-RbohH  were monitored over time at the PT tip and behind the tip when possible, and compared to YC3.60-expressing WT PTs grown and imaged under the same conditions. First, we attempted to study Ca2+ dynamics in anx1 anx2 bulges before bursting, young growing WT PTs, and arrested WT bulges. Bulges of anx1 anx2 never produced a growing tube, and only two out 17 burst during imaging. Interestingly, for both anx1 anx2 bulges that eventually burst, a sudden increase of [Ca2+]cyt was observed  was always observed and quite stable , [Ca2+]cyt was significantly lower than in the WT . But both the tip-focused Ca2+ gradient and the PT growth rate were significantly less steady over time in the rbohH rbohJ double mutant than in the WT, as evidenced by a significantly higher variance  overall lower [Ca2+]cyt levels, and (ii) unstable tip-focused Ca2+ gradients and growth rates. Finally, increasing the external [Ca2+] in the germination medium from 5 mM to 15 mM or 30 mM, significantly decreased the rupture of rbohH rbohJ PTs, while lowering the external [Ca2+] had the opposite effect  fer and rbohC/rhd2 display similar phenotypes with stunted, collapsed, and bursting root hairs, and (ii) that roots and root hairs of fer and one FER-OX line accumulate less and more ROS than WT, respectively rbohH rbohJ double mutants display anx-like phenotypes, i.e., PTs that burst, preventing them from growing to fertilize the female gametophytes  meaningful information about the dynamics of ROS production is still scarce 2O2-selective HyPer sensor expressed in PTs in a rbohH rbohJ NADPH oxidase-deficient mutant background to gain more insights into H2O2 production in tip-growing cells. HyPer activity displayed irregular oscillations at the tip of growing WT PTs  of germinating rbohH rbohJ pollen grains are able to produce longer tubes in vitro that, however, will eventually burst, too. We took advantage of this opportunity to study [Ca2+]cyt dynamics with a good spatial and temporal resolution on growing NADPH oxidase-deficient PTs. First, the tip-focused Ca2+ gradient, visualized by the ratio between [Ca2+]tip/[Ca2+]behind, was clearly visible in growing rbohH rbohJ PTs, confirming that NADPH oxidases are not required to generate the Ca2+ gradient. However, unlike steadily growing WT PTs, which maintain a constant Ca2+ gradient over time , owing to their rapid loss of cellular integrity. On one hand, the NADPH oxidase RbohC has been proposed to generate ROS that activate Caver time , rbohH rds later . This wao WT PTs . An incrt and WT . This is2+ gradient is to facilitate and stimulate exocytosis at the site of growth 2+] leads to root hair and PT growth inhibition and CW thickening anx1 anx2, and rbohH rbohJ plants expressing the ANX1-YFP fusion protein, we show that the rate of exocytosis is significantly increased in ANX1-OX PTs compared to controls. In contrast, for some of the rbohH rbohJ mutant PTs that have low calcium levels and an unsteady Ca2+ gradient, the recovery was impaired during our analysis. In agreement, the Ca2+channel blocker LaCl3, which has been shown to trigger the rupture of root hairs One of the many roles proposed for the tip-focused Ca2+-permeable channels for calcium influx to fine tune the tip-focused Ca2+ gradient, which in turn sustains secretion at the apical tip enabling PTs to elongate steadily without a loss of CW integrity. Perturbations of the pathway by over-expressing ANX RLKs would lead to a NADPH oxidase-dependent over-production of ROS and Ca2+ influx at the PT tip, which in turn would increase the secretion rate of membrane and CW materials, progressively leading to growth cessation and membrane invagination. Conversely, disrupting the ANX RLKs or NADPH oxidase would abolish NADPH oxidases-dependent ROS production and impair the opening of ROS-activated Ca2+-permeable channels, thus limiting the cell's ability to buffer [Ca2+]cyt variation that is required to maintain a steady tip-focused Ca2+ gradient. Consequently, the Ca2+ gradient and exocytosis at the PT tip would become erratic and, if not stabilized by compensatory mechanisms, the CW thickness would decrease until turgor pressure would lead to PT rupture. Finally, our model does not exclude that, in parallel to the signaling events described above, NADPH oxidase-dependent ROS and/or Ca2+ could directly alter CW properties, thereby affecting PT tip-growth. To investigate this possibility, direct measurements of the impact of ROS on CW properties during tip-growth would need to be established. We are confident that combining the continuously improving polar growth models and techniques to measure mechanical properties of growing cells Altogether our data are consistent with the following sequence of events: ANX RLKs positively regulate the NADPH oxidases RbohH and RbohJ, possibly through ROP signaling, to periodically produce ROS. Subsequently, ROS activate Cain vitro incubation on solid germination medium, 100 µl of liquid germination medium containing 0.01% Ruthenium red  or 2 µM of either FM4-64 , CM-H2DCFDA , or FDA  were applied for 5 min to PTs, then washed away with fresh dye-free medium before imaging. PTs stained with Ruthenium red were imaged with a Leica DM6000 (Leica Microsystems). PTs stained with either FM4-64, CM-H2DCFDA, or FDA were imaged with a Leica SP2 or SP5 confocal microscope. For FM4-64 stained PTs, the apical PM region was defined as the first 2.5 µm along PM at the apex. A circle (2.5 µm in diameter) 3 µm away from tip was chosen for measurement of apical cytosol intensity. Relative localization of the FM4-64 dye on the PM versus the apical cytosol was calculated to illustrate the degree of FM4-64 internalization. For CM-H2DCFDA and FDA stained PTs, a circle (4 µm in diameter) 3 µm away from tip was chosen to measure apical cytosol intensity. All dye-derived fluorescence intensities were measured using the ImageJ 1.47d software after background subtraction. PTs of different genotypes were all imaged and quantified under the same conditions.After 3 h to 5 h of anx1-2 anx2-2 (complemented line), WT , and rbohH-1 rbohJ-2 backgrounds were used for FRAP analyses with the same imaging and quantification parameters. The apical region of PTs was photobleached using 100% power of a 514-nm laser (Leica SP5), and the recovery of fluorescence was monitored every 4 s in the following 2 min. The apical PM region was defined on the bright-field pictures at every time frame as the first 2.5 µm along PM at the apex, and fluorescence intensities were measured with ImageJ 1.47d software after background subtraction. Relative intensity of PM-localized ANX1-YFP compared with fluorescence before photobleaching was used to quantify the speed of fluorescence recovery. See Growing PTs expressing ANX1-YFP in an rbohH-1 rbohJ-2 expressing either HyPer or YC3.60 were acquired  and quantified (ImageJ 1.47d) in the exact same conditions. For HyPer, fluorescence was acquired with the sequential mode and excitation at 488 nm and emission between 500–540 nm for F488 and excitation at 405 nm and emission between 500–540 nm for F405. Two circular regions of interest , one 0.5 µm, the other 20 µm away from the apex were drawn for measurement of apical cytosol and far behind the tip intensities, respectively, for each single time point of each PT. For YC3.60, excitation was 458 nm then emission 469–501 nm for FCFP and 522–554 nm for FVenus. Two circular ROIs (4 µm in diameter), one 0.5 µm, the other 10 µm away from the apex, were drawn for measurement of apical cytosol and behind the tip intensities, respectively, for each single time point of each PT. All ratiometric measurements, i.e., F488/F405 and FCFP/FVenus, were determined with ImageJ 1.47d and its Ratio ROI Manager plugin after background subtraction. Ratiometric pictures were generated with the plugin Ratio Stack after median filtering. The YC3.60 titration curve  Quantification of pollen germination rate after 5 h of in vitro growth for WT, two and three independent ANX1-YFP and ANX2-YFP T3 complemented lines, respectively, as well as four independent T3 ANX1-YFP and ANX2-YFP over-expression lines. Data are representative of three experiments with more than 150 pollen for each genotype. The corresponding PT length measurements are displayed in in vitro growth for WT, one ANX1-YFP, and one ANX2-YFP T3 complemented lines, as well as three independent T3 ANX1-YFP and ANX2-YFP over-expression lines. Data represent mean values ± standard error of the mean (SEM) of three independent experiments with more than 40 PTs per genotype and experiment. Double asterisks indicate statistically significant differences from the WT according to a Student's t test with p<0.01.(TIF)Click here for additional data file.Figure S2ANX1-YFP and ANX2-YFP fusion proteins complement the anx1 anx2 pollen tube rupture phenotype. Quantification of pollen germination rate and PT rupture after 5 h of in vitro growth for WT, anx1-2 anx2-2, anx1-2/anx1-2 anx2-2/ANX2, 6, and 5 independent T1 lines of ANX1-YFP in anx1-2/anx1-2 anx2-2/ANX2 and ANX2-YFP in anx1-1/anx1-1 anx2-1/ANX2, respectively, as well as two and three independent T3 homozygous ANX1-YFP in anx1-2 anx2-2 and ANX2-YFP in anx1-1 anx2-1 complemented lines, respectively. More than 150 pollen were analyzed per genotype.(TIF)Click here for additional data file.Figure S3ANX RLK over-expression triggers plasma membrane invagination. (A) Median plane confocal image of an ANX1-YFP over-expressing PT, in which the apical membrane grows inwards. Filters are indicated on the left. Scale bar = 5 µm. (B) Single plane confocal image of plasma membrane invagination in an ANX1-YFP over-expressing pollen grain.(TIF)Click here for additional data file.Figure S4BFA treatment disrupts the enrichment of ANX1-YFP at the apical plasma membrane. (A) Representative median plane sections of anx1 anx2 complemented PTs expressing ANX1-YFP with (right) or without (left) BFA treatment. Note that the YFP-derived fluorescence is much weaker in the apical membrane-derived region of interest of the BFA-treated PTs compared to that of non-treated PTs (left). The same regions of interest (ROIs) were used for FRAP experiments. No correlation between relative fluorescence recovery 10 s after photobleaching and original amount of fluorescent protein in the apical plasma membrane (B) nor with PT growth rate (C).(TIF)Click here for additional data file.Figure S5Structure and expression of Rboh genes in Arabidopsis. (A) The genomic organization of the pollen-expressed NADPH oxidase genes RbohH and RbohJ and positions of the rbohH-1, rbohH-3, rbohJ-2, and rbohJ-3 T-DNA insertions. The orientation of the left border sequence of the respective T-DNAs is represented by black arrows. The positions of the primers used to genotype the mutants are indicated. (B) RT-PCR analyses of cDNAs from open-flowers show no RbohH transcripts in the T-DNA insertion lines rbohH-1 and rbohH-3. There are much less or no RbohJ transcripts in the T-DNA insertion lines rbohJ-2 and rbohJ-3, respectively. UBC21 (At5g25760) was used as a control. Amplification was performed for 30 cycles for UBC21 and for 35 cycles for RbohH and RbohJ. (C) Multiple alignments of Arabidopsis Rboh proteins were performed with ClustalW 1.83 and the phylogenetic tree was reconstructed with MEGA4 using the protein sequence parsimony method . Black and grey circles at nodes indicate bootstrap values of more than 900 and between 800 and 900, respectively. The HsNOX5 was used as outgroup. The tree was then combined with the relative gene expression of ArabidopsisRboh family members in various plant tissues according to the Genevestigator microarray database using the Meta-Profile Analysis tool, Anatomy Profile (TIF)Click here for additional data file.Figure S6Distribution of pollen tubes of ANX1-YFP in anx1 anx2, in wild-type (over-expressor line #1 and #4), and rbohH rbohJ backgrounds relative to the time required to recover 80% of the initial fluorescence. Unlike PTs from complemented and over-expressor lines, some rbohH rbohJ PTs expressing ANX1-YFP were not able to recover 80% of the initial fluorescence at the apical plasma membrane.(TIF)Click here for additional data file.Figure S7rbohH rbohJ2DCFDA-derived fluorescence compared to the wild type. pollen tubes display decreased levels of ROS-sensitive CM-H (A) Single median plane images of growing WT and rbohH rbohJ PTs stained with the ROS-sensitive CM-H2DCFDA dye and imaged with the same settings. Scale bar = 5 µm. (B) Single median plane images of growing WT and rbohH rbohJ PTs stained with the ROS-insensitive FDA dye and imaged with the same settings. The scale is the same as in (A). (C) Quantification of ROS-sensitive CM-H2DCFDA-derived fluorescence in a circle with 4 µm diameter at the tip of growing WT and rbohH rbohJ PTs. Data are mean ± standard error of the mean (SEM) of three independent experiments with more than eight PTs per genotype and experiment. Double asterisks indicate significant differences from the WT according to a Student's t test with p<0.01. (D) Quantification of ROS-insensitive FDA-derived fluorescence in a circle with 4 µm diameter at the tip of growing WT and rbohH rbohJ PTs. Data are mean ± SEM of three independent experiments with more than eight PTs per genotype and experiment.(TIF)Click here for additional data file.Figure S82O2-sensitive HyPer activity at the tip of growing wild-type pollen tubes correlates with GFP-RbohH localization.Pulsating H (A) Time-course ratiometric imaging of the tip of growing WT PT expressing cytosolic HyPer and the corresponding histogram (B) displaying the ratio (F488/F405) at the tip (blue line) over 90 s, as well as the PT growth rates (green line). Scale bar = 5 µm. (C) In vitro PT growth assay showing that the GFP-RbohH protein fusion complements the rbohH rbohJ PT bursting phenotype in T1 rbohH-3 rbohJ-3 heterozygous for GFP-RbohH. Left, rbohH-3 rbohJ-3 pollen. Right, pollen of a representative T1 rbohH-3 rbohJ-3 line expressing GFP-RbohH. (D) Representative single median plane images of a normally growing PT of a GFP-RbohH complemented line (left) and an arrested PT of an GFP-RbohH over-expressing line with apical membrane invagination (right) and over-accumulation of CW material (asterisk). The different filters are indicated. Before imaging, PTs were treated for 5 min with germination liquid medium containing FM4-64 (2 µM). Scale bar = 5 µm.(TIF)Click here for additional data file.Figure S92+− sensitive cameleon YC3.60 ratiometric imaging shows that [Ca2+]cyt levels are decreased in Caanx1 anx2 bulges compared to young, growing wild-type pollen tubes but are similar to arrested wild-type bulges. (A) Representative ratiometric images of young growing WT PT, arrested WT bulge, and bursting anx1 anx2 bulge expressing cytosolic YC3.60. On the ratiometric images, the black circles represent the region of interests of 4 µm diameter used for measurements at the PT tip. At time = 76 s, a white arrow indicates a sudden increase of [Ca2+]tip in anx1 anx2 before the bulge actually bursts (black arrow at time = 84 s). Note how external Ca2+ enters the anx1 anx2 bulge once it has ruptured. See also corresponding CFP/FVenus) at the tip of young, growing WT PTs, arrested WT bulges, and anx1 anx2 bulges before rupture (n>15 for each category). Data are shown as the mean of ratios over 90 s±standard deviation (SD). Double asterisks indicate significant differences from the growing WT PTs according to a Student's t test with p<0.01.(TIF)Click here for additional data file.Figure S102+ gradient and pollen tube growth rates are less stable in Tip-focused CarbohH-1 rbohJ-2 than in the wild type. Representative images of growing WT (A) and rbohH-1 rbohJ-2 (B) PTs expressing cytosolic YC3.60 and the corresponding histograms displaying the ratio of [Ca2+]tip/[Ca2+]behind  over 90 s, and the travelled distance of the PT tip (green line). Note how both the tip-focused Ca2+ gradient and the PT growth rate are more stable in WT compared to the mutant. Scale bar = 5 µm. (C) Histogram of the variance of [Ca2+]tip in M2 of WT (green) and rbohH-1 rbohJ-2 (red). The variance of the [Ca2+]tip is significantly elevated in mutant PTs compared to the WT . (D) Histogram in arbitrary units of the ratio of the YC3.60 of [Ca2+]tip/[Ca2+]behind in growing PTs of WT (green) and rbohH-1 rbohJ-2 (red). (E) Histogram of the variance (arbitrary units) of the ratio of [Ca2+]tip/[Ca2+]behind in growing PTs of WT (green) and rbohH-1 rbohJ-2 (red). The variance of the ratio of [Ca2+]tip/[Ca2+]behind is significantly increased in mutant pollen tubes , indicating that the tip-focused Ca2+ gradient is less stable in the mutants than in the WT. (F) Histogram of the variance of the growth rates in (mm/s)2 for WT (green) and rbohH-1 rbohJ-2 (red) PTs. Note that the variance of the growth rates is significantly higher in mutant PTs compared to the WT , indicating that rbohH-1 rbohJ-2 PT growth is unstable compared to PT growth of the WT.(TIF)Click here for additional data file.Figure S112+ partially suppresses pollen tube rupture of External CarbohH rbohJ mutants. (A) Quantification of the germination rate of pollen from WT, rbohH-1 rbohJ-2, and rbohH-3 rbohJ-3 plants on different Ca2+-containing media. Data are mean ± standard error of the mean (SEM) of three independent experiments with more than 150 pollen grains per genotype and experiment. Single asterisks indicate statistically significant differences from the corresponding control at 5 mM [Ca2+] (indicated by #) according to a Student's t test with p<0.05. (B) Quantification of PT rupture from WT, rbohH-1 rbohJ-2, and rbohH-3 rbohJ-3 plants on different Ca2+-containing media. Data are mean ± SEM of three independent experiments with more than 150 pollen grains per genotype and experiment. Single asterisks indicate statistically significant differences from the corresponding control at 5 mM [Ca2+] (indicated by #) according to a Student's t test with p<0.05. (C) Representative images of WT (top) and rbohH rbohJ (bottom) pollen grains grown in vitro for 5 h on 0 (left) and 15 mM [Ca2+] (right). Scale bar = 5 µm.(TIF)Click here for additional data file.Table S1Parameters of fluorescence recovery after photobleaching (FRAP) at the apical plasma membrane of growing pollen tubes for ANX1-YFP in different backgrounds.(DOCX)Click here for additional data file.Table S2Segregation analysis of rboh mutations by PCR-based genotyping or scoring herbicide resistance of the progeny resulting from reciprocal crosses with the wild type (Col-0).(DOCX)Click here for additional data file.Table S3Segregation analysis of rboh mutations by PCR-based genotyping in the progeny after self-fertilization.(DOCX)Click here for additional data file.Table S4Oligonucleotides used in this study.(DOCX)Click here for additional data file.Text S1Supporting protocols.(DOCX)Click here for additional data file.Video S1Time-course imaging of plasma membrane invagination in an ANX1-YFP over-expressing pollen tube that ceased to elongate. Top, bright-field; middle, YFP-derived fluorescence; bottom, FM4-64-derived fluorescence. The focal plane was adjusted between the different frames to focus on the apical membrane growing inwards. Δt = 40 s. Scale bar = 5 µm.(AVI)Click here for additional data file.Video S2Representative time-course imaging of complemented (top) and ANX1-YFP over-expressing pollen tubes (bottom) treated for 5 min with FM4-64 (2 µM). Δt = 0.56 s. Scale bar = 5 µm.(AVI)Click here for additional data file.Video S3Time-course imaging of representative FRAP experiments on two complemented (top) and two ANX1-YFP over-expressing (bottom) pollen tubes. Bleaching time-stamp occurs at t = 0 s. Green arrow time-stamps appear when 80% of the initial fluorescence is recovered in the apical plasma membrane. Note that ANX1-YFP over-expressing PTs grow slower but recover their fluorescence faster that complemented lines. Δt = 4 s. Scale bar = 5 µm.(AVI)Click here for additional data file.Video S42O2-sensitive HyPer.Time-course imaging of growing wild-type pollen tube expressing the cytosolic H Top and middle panels show the fluorescence collected between 500 and 540 nm after excitation at 488 nm and 405 nm, respectively. Bottom panel shows the corresponding ratiometric image (F488/F405). Note the oscillating HyPer activity at the tip of the growing PT. Δt = 3.26 s. Scale bar = 5 µm.(AVI)Click here for additional data file.Video S5Time-course ratiometric imaging of a young, growing wild-type pollen tube, an arrested wild-type bulge, and a bursting anx1 anx2 bulge. Δt = 4 s. The calibration bar is the same as in 2+]tip in anx1 anx2 before the bulge actually bursts (black arrow at time = 84 s).(AVI)Click here for additional data file.Video S6Time-course ratiometric imaging of three growing wild-type (left) and three rbohH rbohJ2+-sensitive YC3.60. pollen tubes expressing the cytosolic Ca Δt = 3.26 s. Scale bar = 5 µm.(AVI)Click here for additional data file.Video S7Time-course imaging of FM4-64 stained wild-type and rbohH rbohJ pollen tubes growing steadily and unsteadily, respectively. Δt = 1 s. Scale bar = 5 µm.(AVI)Click here for additional data file.


The major pathways for mitochondrial Ca2+ transport include mitochondrial Ca2+ uniporter and Na+/Ca2+ exchanger, as well as mitochondrial permeability transition pore (mPTP) under certain pathophysiological conditions. However, it is still unclear if mitochondrial Ca2+ flux can affect the generation of Ca2+ waves and triggered activities in cardiomyocytes.Recent studies have implicated that mitochondria play important roles in intracellular Ca2+ (Cai2+) was imaged in fluo-4-AM loaded ventricular myocytes isolated from mice. Spontaneous SR Ca2+ release and Ca2+ waves (CaWs) were induced in the presence of high external Ca2+ . The protonophore carbonyl cyanide p - (trifluoromethoxy) phenylhydrazone (FCCP) reversibly raised basal Cai2+ levels in the presence, as well as absence of Cao2+, suggesting Ca2+ release from intracellular stores. Mitochondrial membrane potential (ΔΨm) was monitored by TMRM fluorescence. FCCP at 0.01- 0.1 µM, which partially depolarized ΔΨm, increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed after depolarization amplitudes and frequencies, and induction of triggered action potentials. On the contrary, FCCP at higher concentrations (>0.5 µM), which completely dissipated ΔΨm, eliminated CaWs while the basal Cai2+ remained high. The cease of CaWs was most likely due to the reduction of SR Ca2+ content as evaluated by rapid exposure to10 mM caffeine. Blocking sarcolemmal Na+-Ca2+ exchanger by substituting Na+ with Li+ in the perfusant further elevated basal Cai2+ and restored CaWs. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting ΔΨm) or Ru360 , but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca2+ uniporter activator kaempferol.Cytosolic Ca2+ release and uptake control plasma Ca2+ levels and plays an important role in regulation of intracellular CaWs and arrhythmogenesis.Mitochondrial Ca