The importance played by lipid rafts during the entrance of Ebola and Marburg viruses into the cells was previously proposed by Bavari et al. in 2002. In this article, the authors show that this behavior is linked to the membrane fusion step. Therefore, there should be references to the following papers in this article:Bavari S, Bosio CM, Wiegand E, Ruthel G, Will AB, Geisbert TW, Hevey M, Schmaljohn C, Schmaljohn A, Aman MJ (2002). Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses. J Exp Med 195:593-602.Panchal RG, Ruthel G, Kenny TA, Kallstrom GH, Lane D, Badie SS, Li L, Bavari S, Aman MJ (2003). In vivo oligomerization and raft localization of Ebola virus protein VP40 during vesicular budding. Proc Natl Acad Sci U S A 100: 15936-15941.


Recent studies demonstrate that SOC of pulmonary smooth muscle cells (PASMCs) were upregulated by chronic hypoxia which contribute to the enhanced pulmonary vasoconstriction and vascular remodeling. However, the exact role of STIM1 in the development of chronic hypoxic pulmonary hypertension(HPH) remains unclear.Stromal interaction molecule 1 (STIM1) is a newly discovered Ca3H]-thymidine ([3H]-TdR) incorporation were performed to detect PASMCs proliferation. The cell cycle was analyzed by flow cytometry. The SOC-mediated Ca2+ influx was calculated by Ca2+ fluorescence imaging and the nuclear translocation of NFATc3 was determined by immunofluorescence and Western blot analysis of nuclear extracts.In this study we investigated the cellular distribution and expression of STIM1 by immunofluorescence, qRTPCR and Western blotting methods in Wistar rat distal intrapulmonary arteries under normal and chronic hypobaric hypoxic conditions. In vitro, Wistar rat PASMCs were isolated and cultured. PASMCs were transfected with siRNA targeting STIM1 gene by liposome. The expression of STIM1 protein was detected by Western blotting. [2+ influx and inhibited the nuclear translocation of NFATc3 in hypoxic PASMCs.We found that during the development of HPH and the initiation of vascular remodeling, the mRNA and protein expression levels of STIM1 significantly increased in the distal intrapulmonary arteries. Moderate hypoxia significantly promotes PASMCs proliferation and cell cycle progression. Silencing of STIM1 significantly decreased cellular proliferation and delayed the cell cycle progression induced by hypoxia. Silencing of STIM1 also significantly decreased SOC-mediated Ca2+/NFAT pathway and may represent a novel therapeutic target for the prevention of hypoxia pulmonary hypertension.Our findings suggest that chronic hypobaric hypoxia upregulates the expression of STIM1 in the distal intrapulmonary arteries which plays an important role in the hypoxia-induced PASMCs proliferation via SOC/Ca Hypoxia inhibits the Kv channels, leading to cell membrane depolarization, and triggers Ca2+ influx from the L-type Ca2+channels. The increased Ca2+ levels cause pulmonary arterial smooth muscle cells (PASMCs) to constrict and proliferate which contributes to the development of pulmonary hypertension  were prepared in 0.2% gelatin in PBS and were applied to the sections for 1 h at 37°C. Nuclei were stained using DAPI . Images were taken using a confocal laser scanning microscope .2, 10 mM HEPES, and 10 mM glucose. This was followed by 20 min in reduced-Ca2+ PSS (20 μM CaCl2) at room temperature. The tissue was then digested at 37°C for 20 min in reduced-Ca2+ PSS containing collagenase , papain (9.5 U/ml), bovine serum albumin (2 mg/ml), and dithiothreitol (1 mM). Cells were grown in DMEM supplemented with 10% FBS and were passaged at a 1:3 ratio with trypsin treatment. The purity of the PASMCs in the primary cultures was confirmed using the specific mAb against smooth muscle α-actin. Cells that had been passaged 3 to 8 times and were at 80% confluence were used for all experiments.For isolation of the PASMCs, the adventitia of freshly excised distal (>4th generation) intrapulmonary arteries from adult male Wistar rats were removed. Vascular segments were then cut open, and the endothelium was removed by gently scraping the luminal surface of the vessel. Rat PASMCs were cultured as previously described . In brie2) or hypoxic conditions (3% O2) for 24 h.Small interfering RNA (siRNA) targeting STIM1 (siSTIM1) were designed as previously described  and synt2, 1% (v/v) Triton X-100, 1 mM dithiothreitol, 0.2 mM Na3VO4, 0.4 mM phenyl-methylsulfonyl fluoride, 10 μg/ml leupeptin, and 0.2 mM NaF). After a 15-min incubation on ice, lysates were spun for 5 min  at 4°C. Supernatant containing the cytoplasm fraction was saved, and the pellet, containing the nuclear fraction, was washed once with buffer C and resuspended in buffer N  glycerol, 1 mM dithiothreitol, 0.2 mM Na3VO4, 0.4 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 0.2 mM NaF). Nuclear lysates were incubated for 30 min on a rotating platformat 4°C and spun  for 15 min at 4°C. The amount of total protein was determined using a BCA protein assay kit . An equal amount of total protein (30-50 μg) was loaded and separated by SDS-PAGE. The protein was transferred to polyvinylidene difluoride membranes and was then blocked and probed with the appropriate antibodies. Monoclonal Abs against STIM1 (BD Bioscience), Lamin B1 (Santa Cruz) and β-actin (Santa Cruz) or a polyclonal antibody against NFATc3 (Santa Cruz) were used as primary Abs. The membranes were then washed for 15 min 3 times and incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG for 1 h. Bound antibodies were detected using an enhanced chemiluminescence system following the manufacturer’s instructions. Densitometric signals were quantified by Quantity One software.Forty-eight hours after transfection, cells were collected for protein isolation. The cultured cells were washed twice with ice-cold PBS and lysed on ice in RIPA lysis buffer containing freshly added protease and phosphatase inhibitor cocktails. After 15 min of incubation, the cell lysate was collected by centrifuging the cells for 5 min at 4°C. For nuclear and cytoplasmic fractions, adherent cells were washed in PBS, and the cytoplasmfraction was prepared by the addition of buffer C  and suspended in PBS for 30 min on ice. DNA fluorescence was measured by flow cytometry using an EPICS XL cytometer .2+]i in PASMCs was measured using the Ca2+-sensitive fluorescent indicator fluo-4/AM. Cells were loaded with fluo-4 at 37°C for 30 min using a Ca2+-free physiological salt solution (D-HANKS) containing 5 μM fluo-4/AM. The fluo-4-loaded cells were then perfused with a Ca2+-free physiological salt solution containing the following components: 0.5 mM EGTA to chelate residual Ca2+, 5 μM nifedipine  to prevent calcium entry through L-type voltage-operated Ca2+ channels (VOCC), and 10 μM cyclopiazonic acid  to deplete SR Ca2+ stores. The [Ca2+]i was determined before and after the restoration of extracellular [Ca2+] to 2.5 mM. SOCE was evaluated by measuring the peak increase in [Ca2+]i caused by the restoration of extracellular Ca2+ in the continued presence of nifedipine and CPA.The i pathway under hypoxic conditions  i response to the restoration of extracellular Ca2+. These findings are consistent with those of other smooth muscles [In addition, we used STIM1-specific siRNA to silence the STIM1 expression in rat PASMCs under conditions of hypoxia to investigate the role of STIM1 in the regulation of hypoxia-induced PASMC proliferation. We found that the knockdown of STIM1 expression in PASMCs by RNA interference was highly specific and could be quantitated at the protein level. STIM1 knockdown markedly inhibited SOCE, as measured by the peak [Ca muscles ,31, incl2+-dependent transcription factor. Elevated levels of intracellular Ca2+ increase the activity of the Ca2+-calmodulin-dependent phosphatase calcineurin. Activated calcineurin dephosphorylates multiple serine residues within the regulatory region of the NFAT molecule to induce a conformational change in NFAT that exposes nuclear localisation signals and allows for NFAT nuclear import and the subsequent regulation of gene transcription [2+ influx and the inhibition of the SOC/Ca2+/NFAT pathway was involved in the anti-proliferative effect of sildenafil on PASMCs. These results highlight the important role that the SOC/Ca2+/NFATc3 pathway plays during the development of hypoxic pulmonary hypertension. In this study, we hypothesised that STIM1 knockdown would inhibit the proliferation of PASMCs under hypoxia via the inhibition of the SOC/Ca2+/NFATc3 pathway. By observing the effect of STIM1 knockdown on PASMC SOC-mediated Ca2+ influx and NFATc3 nuclear translocation under hypoxic conditions, we found that hypoxia could increase SOC-mediated Ca2+ influx and promote NFATc3 nuclear translocation. Thus, STIM1 silencing could significantly inhibit SOC-mediated Ca2+ influx and NFATc3 nuclear translocation. These results indicate that STIM1 is an essential regulator of the SOC/Ca2+/NFAT pathway, which plays an important role in hypoxia-induced PASMC proliferation.Nuclear factor of activated T cells (NFAT) is a Cacription . The NFAcription . The NFAcription  reportedcription  reported2+/NFAT pathway, STIM1 may represent a novel therapeutic target for the prevention of hypoxic pulmonary hypertension.In summary, this report presents in vivo and in vitro evidence to indicate that STIM1 may be involved in the development of hypoxic pulmonary vessel remodelling. As hypoxia induces PASMCs proliferation through a STIM1-dependent mechanism and the SOC/CaThe authors declare that they have no competing interests.XH contributed in all the data experiments, analysis and elaborated the figures. JC performed the Western blot, their analysis and contributed to immunofluorescence experiments. YL contributed in qRT-PCR analysis and amelioration of the manuscript. FL and GX contributed in analyzed in vivo measurements and cell culture experiments. YG designed the study, supervised the overall study and wrote the manuscript. All authors have read and approved the manuscript.


In normal epidermis, a Ca2+ gradient exists that is disturbed in psoriatic plaques, favoring impaired keratinocyte proliferation. Several TRPC channels such as TRPC1, TRPC4, or TRPC6 are key proteins in the regulation of high [Ca2+]ex induced differentiation. Here, we investigated if TRPC channel function is impaired in psoriasis using calcium imaging, RT-PCR, western blot analysis and immunohistochemical staining of skin biopsies. We demonstrated substantial defects in Ca2+ influx in psoriatic keratinocytes in response to high extracellular Ca2+ levels, associated with a downregulation of all TRPC channels investigated, including TRPC6 channels. As TRPC6 channel activation can partially overcome this Ca2+ entry defect, specific TRPC channel activators may be potential new drug candidates for the topical treatment of psoriasis.Psoriasis is a characteristic inflammatory and scaly skin condition with typical histopathological features including increased proliferation and hampered differentiation of keratinocytes. The activation of innate and adaptive inflammatory cellular immune responses is considered to be the main trigger factor of the epidermal changes in psoriatic skin. However, the molecular players that are involved in enhanced proliferation and impaired differentiation of psoriatic keratinocytes are only partly understood. One important factor that regulates differentiation on the cellular level is Ca Psoriasis is a common chronic immune skin disease that affects more than 25 million people in North America and Europe 2+ ([Ca2+]ex) increases substantially from the basal over the spinous to the granular layer of the epidermis. As the calcium gradient is thought to represent an important physiological mechanism regulating keratinocyte maturation, defects in this system could easily explain altered proliferation and differentiation of psoriatic keratinocytes. However, the findings of Menon and Elias have so far not been reproduced by others.A first hint was reported nearly 20 years ago by Menon and Elias who described a defect of the epidermal calcium gradient in psoriatic skin 2+ gradient in the epidermis, the differentiation and proliferation of isolated keratinocytes in vitro is regulated by an increase in intracellular Ca2+ ([Ca2+]i) via both Ca2+ release from intracellular stores and Ca2+ influx mechanisms. High extracellular Ca2+ concentrations ([Ca2+]ex) activate the Ca2+ sensing receptor (CaR), a G-protein-coupled receptor 3) and diacylglycerol (DAG) 3 as ligand of IP3 receptors induces release of Ca2+ from the endoplasmic reticulum (ER) and consecutive Ca2+ influx known as “store operated calcium influx” (SOCE). DAG directly activates members of the canonical transient receptor potential (TRPC) channel family and contributes to receptor operated calcium influx (ROC). Among the superfamily of TRP channels, TRPC channels have been suggested to be involved in SOCE, ROC and to play a key role in cell differentiation Similar to the Ca2+ influx 2+]i and differentiation 2+]ex. In addition, TRPC1, TRPC5, TRPC6 and TRPC7 are upregulated after Ca2+-induced differentiation of gingival keratinocytes TRPC channels can be divided into the TRPC1, TRPC4 and TRPC5 group and the TRPC3, TRPC6 and TRPC7 group, whereof DAG directly activates only the latter ones 2+]i homeostasis including the CaR, the release of Ca2+ from the ER or TRPC channels are present in isolated psoriatic keratinocytes and are involved in the observed deficiencies in keratinocyte differentiation. We investigated primary keratinocytes isolated from psoriatic plaques with Ca2+ imaging, western blot analysis, RT-PCR and skin biopsies from psoriasis patients with immunohistochemical staining. We demonstrated substantial defects in Ca2+ influx in psoriatic keratinocytes in response to high extracellular Ca2+ levels, associated with a downregulation of all TRPC channels which are involved in ROC and SOCE, including TRPC6 channels which play a crucial role in keratinocyte differentiation. As TRPC6 channel activation could partially overcome this Ca2+ entry defect in psoriasis keratinocytes, specific TRPC6 channel activators e.g. out of the phloroglucinol class may be potential new drug candidates for the topical treatment of psoriasis.Therefore, we decided to test whether changes in [Ca2+]ex (2 mM) and compared the increase in [Ca2+]i of psoriatic keratinocytes and hpK. The resulting Ca2+ influx was strongly reduced in psoriatic keratinocytes -Ca2+ ATPase and was used to empty Ca2+ stores from ER 2+-free medium raised slightly the [Ca2+]i in both keratinocyte types  and hpK (61.2±8.8 nM).To test if TRPC channels, as downstream targets of CaR, might also be involved in impaired Ca [Ca2+]i  was sign [Ca2+]i . These f2+ response to high [Ca2+]ex by analyzing CaR mRNA levels in psoriatic keratinocytes isolated from 6 patients in comparison to 6 different hpK . TRPC1 was mainly expressed in stratum basale of the epidermis in skin biopsies from healthy controls and, to a somewhat lesser extent in lesional and lesional psoriatic skin  on the expression of the differentiation markers K10 and TGM1 and on cell proliferation was investigated. Hyperforin significantly increased the expression of the early differentiation marker K10 and the late differentiation marker TGM1 in both psoriatic keratinocytes and hpK . Even moin vitro a well as ex vivo analyses to clarify the pathomechanisms of keratinocyte dysfunction in psoriatic skin. Our data show that the impaired calcium homeostasis in psoriatic keratinocytes leading to defects in differentiation and proliferation is associated with downregulated expression of CaR and TRPC channels. Consequently, in calcium imaging experiments, psoriatic keratinocytes showed a reduced calcium influx after stimulation with high [Ca2+]ex. TRPC1, TRPC4, TRPC7 and TRPC6 have been implicated in the calcium-induced differentiation in vitro2+ influx mediated by the TRPC6 activator hyperforin was significantly impaired in psoriatic keratinocytes. A reduced [Ca2+]i in psoriasis keratinocytes has also been reported by Karvonen et al. (2000) following stimulation with thapsigargin, which has been explained by reduced Ca2+ release from the ER 2+]ex. We were able to reproduce and to extend these findings indicating the primary role of defects in Ca2+ entry mechanisms such as SOCE as well as ROC.Intracellular calcium measurements, expression analysis and immunohistological staining allowed us to unravel the defects of psoriatic keratinocytes differentiation and the potential molecular elicitors. This study describes findings from 2+]i. In addition, the expression levels of all TRPC channels on mRNA and protein levels are significantly reduced both in cultured psoriatic keratinocytes and psoriasis plaques, as we could detect with RT-PCR and western blot analysis. Our findings of a diminished TRPC1-, TRPC4- and TRPC6-channel expression were confirmed by immunohistological stainings of punch biopsies from psoriasis plaques. This approach already demonstrated a downregulation of TRPC channels in non lesional skin which is even more pronounced in lesional skin from the same patient and healthy controls. This provides a more plausible explanation for the link between defects in calcium homeostasis and disturbed differentiation of psoriatic keratinocytes. Up to now, only external elicitors like a loss of the calcium gradient, abnormal barrier function or enhanced production of the cytokines IL-20 or IL-22 in T cells (and consecutively in keratinocytes) have been hypothesized to play a role in the altered differentiation and proliferation in psoriatic skin in vivo environment which is in agreement with many other studies (see the comprehensive review by Tschachler) in vivo. Karvonen et al. (2000) postulated an inherited defect in calcium metabolism because the altered Ca2+ influx after store depletion was still present up to the fifth passage of cultured psoriatic keratinocytes and could be also detected in keratinocytes isolated from non lesional skin. These assumptions are supported by the findings that keratinocytes isolated from lesional and nonlesional skin equally appear to be “diseased” and that additional stressors such as cytokines can trigger the formation of psoriatic plaques As our results implicated a defect in CaR and/or TRPC channels, we examined the expression pattern of CaR and TRPC channels in cultured psoriatic keratinocytes compared to control human keratinocytes. By RT-PCR a slightly reduced expression of CaR could be detected which however seems to be too small to solely explain the pronounced changes in [Ca2+]i but also at least partially overcomes the intrinsic defect of maturation. Hyperforin increased keratinocyte differentiation as indicated by the enhanced expression of the differentiation markers K10 and TGM1 in psoriatic keratinocytes. However, hyperforin is not able to restore the defect completely to hpK control levels. Furthermore, hyperforin decreased proliferation in psoriasis keratinocytes and hpK to similar extent which might be another pathophysiologically important aspect. Furthermore, TRPC channels are discussed to be involved in Darier's disease and atopic dermatitis The hypothesis that TRPC channels might be a novel target for the treatment of psoriasis is supported by our findings that specific TRPC6 activation by hyperforin not only elevates  and secondary antibodies  conjugated to horseradish peroxidase and processed for visualization by ECL™ Reagent . The antibody for Glyceraldehyde-3-phosphate dehydrogenase   served as loading control. Relative intensities of the bands were quantified by densitometry with ChemiDoc XRS System and Quantity One Software (Bio-Rad).2+ concentration [Ca2+]i, measurements in single cells were carried out using the fluorescence indicator fura-2-AM in combination with a monochromator-based imaging system  attached to an inverted microscope . For [Ca2+]i measurements psoriatic keratinocytes and hpK were loaded with 4 µM fura-2-AM (Invitrogen) and 0.01% Pluronic F-127 (Invitrogen) for 30 min at room temperature in a standard solution composed of 138 mM NaCl, 6 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5.5 mM glucose, and 10 mM HEPES (adjusted to pH 7.4 with NaOH). Cover slips were then washed in this buffer for 20 min and mounted in a perfusion chamber on the microscope stage. To measure [Ca2+]i in Ca2+-free environment cells were loaded with fura-2-AM and then washed in Ca2+-free standard solution. For all fluorescence experiments, fluorescence was excited at 340 and 380 nm. After correction for background fluorescence, the fluorescence ratio F340/F380 was calculated.The intracellular CaQuantification of cell proliferation was determined by a non-isotopic immunoassay kit , based on the measurement of BrdU incorporation during DNA synthesis. The assay was carried out according to the product instruction manual.t-test (two-tailed) was used. Data are expressed as mean ± SEM.In addition to Microsoft Office Excel, GraphPad PRISM™ (Version 3.0) was used for statistical analyses and to create the graphs. For statistical analyses, an unpaired Student's Data S1(0.17 MB TIF)Click here for additional data file.


Retroviruses and many other enveloped viruses usurp the cellular ESCRT pathway to bud from cells. However, the stepwise process of ESCRT-mediated virus budding can be challenging to analyze in retroviruses like HIV-1 that recruit multiple different ESCRT factors to initiate budding.In this study, we characterized the ESCRT factor requirements for budding of Equine Infectious Anemia Virus (EIAV), whose only known direct ESCRT protein interaction is with ALIX. siRNA depletion of endogenous ESCRT proteins and “rescue” experiments with exogenous siRNA-resistant wild type and mutant constructs revealed budding requirements for the following ESCRT proteins: ALIX, CHMP4B, CHMP2A and VPS4A or VPS4B. EIAV budding was inhibited by point mutations that abrogate the direct interactions between ALIX:CHMP4B, CHMP4B:CHMP2A, and CHMP2A:VPS4A/B, indicating that each of these interactions is required for EIAV budding. Unexpectedly, CHMP4B depletion led to formation of multi-lobed and long tubular EIAV virions.We conclude that EIAV budding requires an ESCRT protein network that comprises EIAV Gag-ALIX-CHMP4B-CHMP2A-VPS4 interactions. Our experiments also suggest that CHMP4B recruitment/polymerization helps control Gag polymerization and/or processing to ensure that ESCRT factor assembly and membrane fission occur at the proper stage of virion assembly. These studies help establish EIAV as a streamlined model system for dissecting the stepwise processes of lentivirus assembly and ESCRT-mediated budding. The ESCRT pathway is therefore mobile machinery that is targeted to different cellular membranes to mediate different “reverse topology” membrane fission events.Many enveloped viruses usurp the cellular ESCRT pathway to bud from cells. The ESCRT pathway also mediates membrane fission during vesicle formation at the multivesicular body (MVB), abscission of the intercellular bridge, and shedding microvesicle formation at the plasma membrane (reviewed in -8). All S. cerevisiae[S. cerevisiae has more than 20 ESCRT-associated proteins, however, and it has therefore proven useful to divide them into essential factors that are required for MVB sorting, and non-essential accessory factors that appear to modulate or regulate core protein functions (reviewed in reference [S. cerevisiae MVB sorting pathway are: 1) ESCRT-0 (a two protein complex), 2) ESCRT-I (a four protein complex), 3) ESCRT-II (a three protein complex), 4) Vps20p/CHMP6, Snf7p/CHMP4A/B/C, Vps24p/Did3p/CHMP3, and Vps2p/Did4p/CHMP2A/B , and 5) Vps4p/VPS4A/B.ESCRT factors assemble in a stepwise fashion in which “early-acting” factors bind site-specific adaptors and then recruit the “late-acting” factors that mediate membrane fission and ESCRT factor recycling. The ESCRT pathway was first identified through genetic studies of MVB sorting in erevisiae-13, and erevisiae, the abieference ). AlthouS. cerevisiae MVB protein sorting [TSG101 subunit [CHMP6, leading to recruitment of the ESCRT-III subunits through direct, ordered interactions between Vps20p/CHMP6, Snf7p/CHMP4A-C, Vps24p/Did3p/CHMP3, and Vps2p/Did4p/CHMP2A-B[CHMP2A/B and Snf7p/CHMP4A/B/C, also recruit the Vps4p/VPS4A/B AAA ATPase, using two different types of C-terminal tail motifs  to bind Vps4 MIT domains [S. cerevisiae include three ESCRT-III-like proteins: Vps44p/CHMP1A/B, Ist1p/IST1, and Vps60p/CHMP5, which interact with both core ESCRT-III subunits and with Vps4p [LIP5, which binds both Vps60p/CHMP5[VPS4A/B, and stimulates enzyme assembly and ATPase activity [ALIX, which binds and stabilizes the Snf7p/CHMP4A/B/C filaments, and recruits the deubiquitinating enzyme, Doa4p/UBPY[The core ESCRT factors are recruited sequentially to sites of  sorting . The ESC subunit . ESCRT-I subunit ,17. ESCR/CHMP2A-B-21. The /CHMP2A-B,22-31, a domains -35. Vps4 domains ,37, resuth Vps4p -42; Vta160p/CHMP5,44 and Vactivity -48; and oa4p/UBPY,49.nL” late assembly domains function by binding ALIX; and “PPXY” late assembly domains function by binding members of the ESCRT-associated NEDD4 family of ubiquitin E3 ligases. Recent studies, particularly of HIV-1, make it clear that these initial interactions ultimately result in the recruitment of downstream ESCRT-III and VPS4 proteins, which carry out the membrane fission step [Although the core yeast ESCRT components and their mechanistic functions are largely conserved across eukaryotes, there are likely to be important differences in the way the pathway is used to perform distinct membrane fission reactions, particularly in processes like enveloped virus budding that do not occur in yeast. ESCRT-mediated enveloped virus budding has been most intensively studied for retroviruses, particularly HIV-1 . ion step -58. HowenL motifs that bind directly to TSG101/ESCRT-I and ALIX, respectively [One challenge in dissecting how the ESCRT pathway functions in retrovirus budding is that mammalian cells express a large number of isoforms of the different classes of ESCRT factors, including at least 12 distinct subunits of the ESCRT-III family. Adding to this complexity is the fact that the Gag proteins from many retroviruses contain multiple late assembly domains that can bind and recruit different early-acting ESCRT factors. For example, HIV-1 Gag contains both P(S/T)AP and YPXectively -62. Thesectively ,64, and ectively ,65, and ectively ,66,67. Iectively ,68-72. Tectively ,74, indiectively ,76, and ectively . The funS. cerevisiae[We used siRNA depletion experiments to test the requirements for all of the human ESCRT factors that correspond to core ESCRT factors for MVB sorting in erevisiae: ESCRT-INL4-3 or EIAV, together with either control siRNAs , or with siRNAs that targeted ALIX (lane 3), TSG101 (lane 4), CHMP2A and B (lane 5), CHMP4A and B (lane 6), or VPS4A and B (lane 7). Viral titers were measured in single-cycle infectivity assays (panel 1), and virion release was analyzed using western blotting to quantify the levels of virion-associated CA proteins released into the culture supernatant . Western blots of soluble cell extracts were also performed to visualize Gag protein expression and processing (panel 3), GAPDH levels , and the efficiency of target protein depletion (bottom panels). In every case, the siRNA treatment reduced target protein levels more than 10-fold.Requirements for the remaining core ESCRT proteins in HIV-1 and EIAV release and infectivity are compared in Figure As expected, our results for the HIV-1 control . These results are consistent with previous reports that the EIAV p9Gag polypeptide contains a functional YPDL late domain that recruits ALIX, but lacks a TSG101 binding site [As shown in Figure ing site ,63,68-72Gag reproducibly increased when CHMP4B was depleted, either alone or in conjunction with other CHMP4 proteins , denoted “CHMP2-”, compare lanes 6 and 4, and see ref. [Analogous experiments were used to test the functional requirements for CHMP4 and CHMP2 in EIAV release Figure . As showsee ref. ). Similasee ref. ). These see ref. ,56.The VPS4 protein requirements for EIAV release were also tested using functional rescue experiments. As shown in Figure VPS4 proteins can bind ESCRT-III substrates through a second type of MIT-binding motif (termed MIM2) ,78. Thisreduced the viral titer 30-fold, but increased virion-associated Gag protein levels 4.7-fold . Gag processing was also less complete in virions released from cells lacking CHMP4 proteins as compared to the control case. To characterize these phenomena further, we used transmission electron microscopy (EM) to visualize the morphology of the cell-associated EIAV virions produced from control 293T cells, or from cells depleted of ALIX, CHMP2 or CHMP4 proteins  spherical, mature, cell-free virions, 2) spherical, immature, cell-free virions, 3) spherical budding virions, and 4) multi-lobed or tubular virions  or CHMP6 (the only ESCRT-III protein known to bind ESCRT-II). Hence, EIAV, and presumably also some SIV strains [Our experiments confirm that HIV-1 and EIAV differ in their requirements for TSG101, consistent with the lack of a known TSG101 binding site in EIAV p9 strains ,74, use Gag binds ALIX [Gag through the Bro1 domain [Protein-protein interactions in the EIAV budding pathway are summarized in Figure nds ALIX , with thnds ALIX . ALIX cands ALIX , dimeriznds ALIX , bind NC1 domain -84, and 1 domain ,86, alth1 domain , and thiS. cerevisiae, where the apparent ALIX homolog, Bro1p, was reported to function downstream of Snf7p (CHMP4) [ALIX appears to perform analogous “early” assembly functions in a series of other mammalian ESCRT-dependent processes, including abscission -90, MVB  (CHMP4) . A recen (CHMP4) , and so in vitro[in vitro[in vitro[in vitro[in vitro HIV-1-based ESCRT assembly system [S. cerevisiae) suppresses the temperature- and concavanine-sensitivity induced by deletion of Vps24p/Did3p (the CHMP3 homolog) from S. cerevisiae[P. falciparum and E. histolytica appear to have CHMP4 and CHMP2 homologs, but lack CHMP3 homologs [CHMP4B helps to recruit CHMP2A. CHMP4B and CHMP2A can interact directly in vitro,57, and [in vitro,56. A su[in vitro, appears[in vitro, and pererevisiae. Similarhomologs .It is not yet clear why CHMP2A is the primary CHMP2 isoform required for EIAV release from 293T cells  with 10% FCS. HeLa-TZM cells were obtained through the AIDS Research and Reference Reagent Program.siRNA (19 nt + d(TT) overhangs) were designed using the Dharmacon siDESIGN Center (Thermo Fisher Scientific Inc.) and were synthesized by the University of Utah core facility. siRNA sequences are provided in Additional file 5 cells/well in 6-well plates; t = 24 hr, transfection with 10 nM siRNA and 7.5 μl lipofectamine RNAimax (Invitrogen); t = 48 hr, media change (2 ml) and co-transfection with 10 nM siRNA, with control vector or siRNA-resistant expression construct (0.7 μg), and with control vector and viral expression vector(s)  48 h after the second siRNA transfection, washed three times for 10 min with 50 mM cacodylate buffer, stained with a 2% OsO4 solution for 1 h, rinsed three times for 10 min with water, and stained with a 4% uranyl acetate solution for 30 min. Samples were dehydrated with a graded ethanol series, then in acetone, and embedded in epoxy resin Embed-812 (Electron Microscopy Sciences). Thin sections (80–100 nm) were picked up on copper grids, stained for 20 min on drops of saturated uranyl acetate, rinsed with water and then stained for 10 min on drops of Reynolds’ lead citrate. Electron micrographs were collected on a Hitachi H-7100 transmission electron microscope at an accelerating voltage of 75 kV, equipped with a Gatan Orius sc1000 camera.Virus-producing 293T cells were pelleted at low speed, fixed with 2.5% glutaraldehyde/1% paraformaldehyde in cacodylate buffer (0.1 M sodium cacodylate (pH 7.4), 35 mM sucrose, 4 mM CaClTwo independent experiments were performed, and a total of 28 cells with associated EIAV virions were counted for each condition and scored for the presence of: mature virions, immature virions, budding virions  and multi-lobed or tubular virions (virions with multiple lobes or long tubular structures). Examples of these different phenotypes are given in Figure The authors declare that they have no competing interests.VS performed the experiments, and VS and WIS analyzed the data and wrote the manuscript. Both authors read and approved the final manuscript.Supplemental Text and References.Click here for fileEffects of CHMP2 and CHMP4 depletion on EIAV release and infectivity.Click here for fileDepletion of EAP20, CHMP3 or CHMP6 does not significantly affect EIAV release or infectivity.Click here for fileRelease of EIAV virions from control 293T cells.Click here for filesiRNA sequences used in this study.Click here for fileExpression vectors used in this study.Click here for file


The professional development of under-represented faculty may be enhanced by mentorship, but we understand very little about the mechanisms by which mentoring brings about change. Our study posed the research question, what are the mechanisms by which mentoring may support professional development in under-represented groups?The study aims to: (i) to pilot a mentoring scheme for female academics; (ii) to compare various health-related and attitudinal measures in mentees at baseline, 6 months, and 1 year into the mentoring relationship and, (iii) to compare pre-mentoring expectations to outcomes at 6 months and 1 year follow-up for mentees and mentors.Female academic mentees were matched 1:1 or 2:1 with more senior academic mentors. Online surveys were conducted to compare health-related and attitudinal measures and expectations of mentoring at baseline with outcomes at 6 months and 1 year using paired t-tests and McNemar's test for matched cohort data.N = 46 mentoring pairs, 44 (96%) mentees completed the pre-mentoring survey, 37 (80%) at 6 months and 30 (65%) at 1 year. Job-related well-being (anxiety-contentment), self-esteem and self-efficacy all improved significantly and work-family conflict diminished at 1 year. Highest expectations were career progression , increased confidence , development of networking skills , better time-management  and better work-life balance . For mentees, expectations at baseline were higher than perceived achievements at 6 months or 1 year follow-up.For mentors (N = 39), 36 (92%) completed the pre-mentoring survey, 32 (82%) at 6 months and 28 (72%) at 1 year. Mentors' highest expectations were of satisfaction in seeing people progress , seeing junior staff develop and grow , helping solve problems , helping women advance their careers  and helping remove career obstacles . Overall, gains at 6 months and 1 year exceeded pre-mentoring expectations.This uncontrolled pilot study suggests that mentoring can improve aspects of job-related well-being, self-esteem and self-efficacy over 6 months, with further improvements seen after 1 year for female academics. Work-family conflict can also diminish. Despite these gains, mentees' prior expectations were shown to be unrealistically high, but mentors' expectations were exceeded. In academic institutions women advance more slowly in rank than men, hold fewer leadership positions, receive lower salaries and obtain fewer research grants ,2. AlthoThere is some evidence from the USA that introducing mentoring schemes improves career trajectories for female academics ,6 as welThe existing evidence base for mentoring is limited . Most stTherefore we introduced a pilot mentoring scheme for female academics at the Institute of Psychiatry, King's College London, UK. We undertook a comprehensive longitudinal evaluation of our scheme collecting data from both mentee and mentor at baseline, 6 months and 1 year. We aimed to: (i) pilot the mentoring scheme, (ii) evaluate the health-related and attitudinal benefits mentees gained from the experience in terms of self-esteem, self-efficacy, job-related wellbeing, work interference with family and job satisfaction and (iii) compare both mentee and mentor pre-mentoring expectations, to achievements and gains at 6 months and 1 year follow-up. Our study used quantitative measures in an academic setting to ask the research question, what are the mechanisms by which mentoring may support professional development in under-represented groups?The pilot mentoring scheme for female academics was launched at the Institute of Psychiatry, King's College London, UK, in July 2008 as part of a series of interventions across the University (The Womens' Advancement Initiative). The Institute of Psychiatry is a school of King's College London, with approximately 1,000 members of staff, 42% of whom are female academics, many of whom have clinical as well as academic roles. In 2007, when the scheme was set up, only 26% of professors were women, compared to 61% of lecturers . The scheme was developed during a consultation process to establish best practice which involved a series of interviews with key stakeholders/opinion leaders who were involved in the setting up and delivery of formal mentoring schemes in the UK and USA and/or positive gender action schemes (further details available from the authors upon request).Mentees self-selected to be part of the scheme, from a potential participant pool comprising all female academic staff of senior lecturer level or below who had a contract with the Institute of Psychiatry of at least 1 year. Potential mentees were identified from staff lists and were sent information packs and invitations for 'taster lunches'.Mentors  were recruited simultaneously, by invitation (on the basis of recommendations from junior colleagues), by self-sign up after publicity of the scheme, or by specific nomination by an individual mentee at the point of recruitment into the scheme. Strict selection criteria were not applied as the most important factor was motivation to participate in this pilot study.Mentees were matched with a mentor who they had either nominated, by browsing 'pen portraits' of available mentors, or were matched in a 'clinic' run by the scheme organiser (ACI) - in which 17 potential mentees were interviewed and matched to appropriate mentors according to their seniority level and requirements from mentoring.http://www.gptrainingconsultants.com). This consisted of an initial training session in key skills of developmental mentoring "• "the mentoring program has made me more assertive to ask for more in the workplace"(ii) Support and encouragement• ".. .encouragement to do some new and challenging things for the benefit of my career"• "invaluable to have someone outside my section, but within my department who has some insight into the frustrations of being mid-career and is able to give some useful support"(iii) Space to reflect on careers and goals• "... support, encouragement, a space for thinking and talking about work issues and my career and thinking about what I want, practical strategies and information"For mentors, the five highest expectations were of satisfaction in seeing people progress 25/36; 69%), seeing junior staff develop and grow , helping to solve problems , helping women advance their careers  and helping to remove obstacles to their careers . However, when measuring perceived gains at 6 months and 1 year, the proportion of mentors who endorsed the statement: 'a good relationship with my mentee', was ranked as one of the highest gains , although it was not a high expectation at outset . In most cases, gains and expectations were similar but there were sometimes gains at 6 months and 1 year that had not been expected at the pre-mentoring stage. This was especially true for having a good relationship with the mentee and for helping to solve problems  Being able to give something back• "... to pass on some of the insights that I have gained from my experiences at the Institute of Psychiatry to the next generation"(ii) To see the Institute of Psychiatry from a different perspective• "... learning about a different working culture and different challenges in another Institute of Psychiatry department (and being grateful that these factors don't seem so bad within my own!)"(iii) Providing support and seeing mentee develop• "... knowing that my mentee is now feeling better about the issues she came with and is more optimistic about her career progression than when I first met her"(iv) Reflection on own career and skills• "I have been very impressed with my mentee's resilience in the face of some very major issues in her life. This has made me reflect on some of my own ways of dealing with challenges"Self-esteem, self-efficacy and job-related well-being (anxiety-contentment) improved significantly with mentoring. Work-family conflict had diminished at 1 year follow-up. Improved confidence and assertiveness, receipt of support and encouragement and the space to reflect on careers and goals emerged as important benefits. However neither the mentee job satisfaction scores increased nor was the pre-mentoring expectation of 'career progression' achieved by 1 year. For mentors the perceived gains at 6 months and 1 year often exceeded pre-mentoring expectations; mentoring yielded some unexpected rewards.Our results regarding a significant improvement in self-esteem at 6 months and 1 year are consistent with Ragins and colleagues examination of the mentee perspective of mentoring in a management setting, which found that mentored individuals had significantly higher levels of organizational based self-esteem than their non-mentored counterparts . AlthougSelf-efficacy is the belief that one has the capabilities to execute the courses of actions required to manage a prospective situation and is the central construct in Bandura's social cognitive theory . It is tBoth women's global self-esteem  and task-specific self-efficacy (which captures the self-assessed ability to perform a certain task) have been shown to be predictors of self-limiting behaviour in certain leadership tasks . In a stWe noted that there was no difference between job-related depression-enthusiasm at 6 months or 1 year compared to pre-mentoring, whereas there was an improvement in the anxiety-contentment scale. Mentoring may be a factor in improving contentment at work, as suggested by a review concerning 'authentic happiness' in dentists, as a result of mentoring within professional development programmes . There mThe work interference with family scale was used to understand whether mentors play a role in influencing mentees' perceptions of the work-family interface. It appeared by 1 year that there was some overall reduction in the level of perceived conflict. In a field study of 502 male and female graduates from American business management courses, who were either involved in a committed relationship or had children living at home, it was found that individuals with mentors reported significantly less work-family conflict than those without mentors . HoweverOur inability to demonstrate an improvement in job satisfaction was unsurprising given that at the time the mentoring scheme was introduced and evaluated at the Institute of Psychiatry, it was in the context of considerable institutional and national economic uncertainty with a local freeze on posts, and a 'Sustainability Review' at the Institution which involved redundancies. The fact that despite this, there were improvements in many of our outcome measures suggests that at an individual level, mentoring was still of benefit for mentees.The high levels of mentee expectations which we found at the pre-mentoring stage have also been reported in some cases studied by Wildman et al  of experIt is noteworthy that in many areas, the mentors appeared to gain more from their experiences than they expected. Clutterbuck notes that powerful often unpredicted benefits exist for mentors and these often prompt reassessment of their own views and leadership style . A modifOur study has several limitations. First, it was an uncontrolled pilot study, aimed at feasibility and establishing any effects. The lack of any control group means that our results need to be seen as indicative only. Second, the limited numbers of mentees and mentors in this pilot study reduces the power of any statistical analysis and to protect staff anonymity, mentee demographic data  were not collected. Third, there was some missing data at each time point which means that the paired t tests and matched McNemar's tests were based only on complete data. Fourth, all the mentors and mentees were self-selected and their interest in mentorship may have skewed the results towards a more favourable estimate of the personal gains and achievements from mentoring, and of higher and more unrealistic expectations. Fifth, the spectrum of type and rank of academics involved in this small study was broad, and the results are not representative of one particular occupational group or demographic, although all staff worked in a specialist academic mental health institution. Sixth, objective outcomes of career progression (such as publishing research papers or obtaining grants) were not included because of the time scale.This was a feasibility pilot study of mentoring. To determine the true effect of mentoring in an institution a randomised controlled trial would be an important next step.In any trial which builds on this pilot work it will be important to also track objective outcome measures such as mean number of publications, change in H-index, number of research grants awarded and promotion within academia, as indicated by Sambunjak et al  in theirThe potential benefits of academic mentoring for women are important. This pilot study has indicated that mentoring could contribute to women's personal and professional development. The study was also able to begin to demonstrate the mechanisms involved. Rigorous evaluation of mentoring as an intervention would be the next step for institutions which want to help retain and develop the careers of their academic staff, particularly their women academics.The authors declare that they have no competing interests.RD reviewed the literature, undertook the statistical analyses, interpreted the data and drafted the manuscript which was critically revised by the other authors. SLH participated in the design, collected the data and was responsible for the coordination of the study. EK helped conceive and design the study, protocol and interpret the data. DG was involved in the design of the study, and the choice of measures, and commented on the manuscript. NTF and ACI conceived of and designed the study, wrote the protocol and participated in interpretation of the data. NTF assisted with the statistical analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6920/11/13/prepubAppendix. Questions used as quantitative measures.Click here for file


Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with a dismal long-term survival. Novel therapeutic agents are therefore urgently required to improve outcomes. AML is highly heterogeneous at the molecular, cellular and clinical level, however common themes have recently emerged, including transcriptional dysregulation and alteration of epigenetic control. Indeed, deep sequencing efforts have outlined recurrent mutations in a number of epigenetic regulators in AML. This observation has potential clinical implications for, whilst the genome is fixed, the epigenome is dynamic, making it a potentially attractive target for therapeutics. This talk will outline the clinical and biological background to AML and will also provide context for targeting epigenetic readers.The bromodomain and extra-terminal (BET) proteins are a family of bromodomain-containing epigenetic readers. They bind to acetylated lysines in histones and other proteins and interact with a number of transcriptional regulators during transcriptional commitment, initiation and elongation. Recently, inhibitors of the protein-protein interaction between BET proteins and acetylated lysines have been developed, initially as anti-inflammatory immunomodulators. We will outline our rationale for utilising these inhibitors in a highly specific form of acute leukaemia, mixed lineage leukaemia (MLL)-fusion leukaemias, and will briefly show published data justifying this hypothesis. These data will outline the mechanism of action of these inhibitors, demonstrating the removal from chromatin of BET proteins and interacting transcriptional complexes, leading to downregulation of transcription at loci critical for leukaemogenesis. Finally, preclinical studies demonstrating the efficacy of these inhibitors in murine models, cellular models and patient samples of MLL-fusion leukaemia will outline the efficacy and clinical promise of these agents for this aggressive form of leukaemia.The majority of the talk will focus on unpublished data investigating the extension of I-BET therapy to other subtypes of AML. We will demonstrate that many other subtypes of AML are sensitive to growth inhibition with I-BET. This sensitivity appears to be due to the downregulation of a common transcriptional programme across a number different AML subtypes. We will show data predicting that this programme, along with specific recurrent mutations, will form the basis of predictive biomarkers of sensitivity and response to I-BET therapy, thus informing clinical trials. We next focused our attention on AML with mutations of the nucleophosmin (NPM1) gene and describe the mechanism underpinning the profound sensitivity of this common AML subtype. Finally, we will show evidence of preclinical efficacy in murine, cellular and primary patient material models of NPM1c AML.At the end of the talk I will attempt to put these findings into clinical context, briefly summarising the results of BET inhibition in other haematological malignancies and discussing an outline of plans for a phase I clinical trail of BET inhibition in relapsed haem malignancies to be carried out within the UK.


The sustained, non-inactivating current occurred at voltages where voltage-dependent activation and inactivation overlapped (voltage window) and its contribution to basal tone or active tension in larger multicellular blood vessel preparations is unknown at present. This study investigated whether window Ca2+ influx affects isometric contraction of multicellular C57Bl6 mouse aortic segments.Electrophysiological studies of L-type Ca2+ , membrane potential and isometric force were measured in aortic segments, which were clamped at fixed membrane potentials by increasing extracellular K+ concentrations. K+ above 20 mM evoked biphasic contractions, which were not affected by inhibition of IP3- or Ca2+ induced Ca2+ release with 2-aminoethoxydiphenyl borate or ryanodine, respectively, ruling out the contribution of intracellular Ca2+ release. The fast force component paralleled Cai2+ increase, but the slow contraction coincided with Cai2+ decrease. In the absence of extracellular Ca2+, basal tension and Cai2+ declined, and depolarization failed to evoke Cai2+ signals or contraction. Subsequent re-introduction of external Ca2+ elicited only slow contractions, which were now matched by Cai2+ increase. After Cai2+ attained steady-state, isometric force kept increasing due to Ca2+- sensitization of the contractile elements. The slow force responses displayed a bell-shaped voltage-dependence, were suppressed by hyperpolarization with levcromakalim, and enhanced by an agonist of L-type Ca2+ channels (BAY K8644).Intracellular Ca2+ influx via Ca2+ channels which completely inactivate. Ca2+ channels, which did not completely inactivate during the depolarization, initiated a second, sustained phase of contraction, which was matched by a sustained non-inactivating window Ca2+ influx. Together with sensitization, this window L-type Ca2+ influx is a major determinant of basal and active tension of mouse aortic smooth muscle.The isometric response of mouse aortic segments to depolarization consists of a fast, transient contraction paralleled by a transient Ca At 30 and 50 mM K+ these spikes disappeared, but force developed with a fast and slow component. Above 50 mM K+ only a small increase (50–70 mM K+) or even a decrease (90–124 mM K+) of force was observed  were not significantly different from the steady state values measured with repetitive depolarization (vide supra).In the cumulative protocol, two force signals were seen at 15 and 20 mM K+ contractions or to the K+-dose–response relationships in aortic segments -mediated Ca2+ release. Contractions by 2 μM phenylephrine (PE) in the absence of extracellular Ca2+ were significantly reduced by 50 μM 2-aminoethoxydiphenyl borate (2-APB), a blocker of IP3-induced Ca2+ release-contraction curve even more. This suggests that the decrease of force at 90 and 124 mM K+ was not due to a reduction in sensitivity to Ca2+, but was proportional to the window influx of Ca2+ via L-type Ca2+ channels  are expected to affect Ca2+ influx and contraction of the segments. Segments could be hyperpolarized from −60 mV to the K+ equilibrium potential (VK) of −86 mV at 5.9 mM K+ with levcromakalim (200 nM), an opener of ATP-dependent K+ channels , an activator of L-type Ca2+ channels[+ concentrations in the presence of levcromakalim, BAY K8644, or their combination , whereas BAY K8644 caused a shift to lower K+ concentrations (-7.98 ± 1.09 mM). Both effects were fully additive, indicating independent effects of Vm  and L-type Ca2+ channel gating (BAY K8644) on window contractions. Changes of Vnels see. The L-tAY K8644  nM, an achannels,34. When+, levcromakalim caused a glibenclamide (inhibitor of ATP-sensitive K+ channels)-sensitive decline of intracellular Ca2+  and baseline tension , whereas BAY K8644 raised resting intracellular Ca2+ (+0.016 ± 0.008 RU) and force . The BAY effect could be reversed by addition of levcromakalim or nifedipine (data not shown). To illustrate the physiological importance of the window Ca2+ influx for basal contraction of mouse aortic segments, the external K+ concentration was changed to obtain depolarizations or repolarizations within the physiological range of Vm for VSMCs . These data provide further evidence for the importance of window Ca2+ influx within the physiological range of Vm or K+ concentrations[At normal extracellular Ktrations,36.2+ via L-type Ca2+ channels. Thereby, both Ca2+ influx and contraction depended on the amplitude of depolarization  and on the resting potential of the VSMC . At resting membrane potentials, elevation of extracellular K+ above 10–20 mM caused biphasic contractions and Ca2+ signals. Although the relationships between intracellular Ca2+ and force appeared to be complex and sometimes non-linear (slow component), we demonstrated that the fast, phasic force component was related to a transient Ca2+ influx, presumably via a population of L-type Ca2+ channels which activated and completely inactivated during the depolarization. On the other hand, the slow, tonic force component displayed a bell-shaped voltage (K+)-dependence and could be attributed to voltage-dependent, “steady state” Ca2+ influx via a population of L-type Ca2+ channels. These channels did not completely inactivate during sustained depolarization and gave rise to a window contraction. In addition to the Ca2+ influx via both populations of L-type Ca2+ channels, a time-dependent Ca2+ sensitization contributed to the depolarization-induced contractions of the mouse aorta.The present study showed that the main determinant of depolarization-induced contractions of the mouse aorta was the influx of extracellular Ca+ were mainly due to influx of extracellular Ca2+ via L-type Ca2+ channels in the mouse aorta. Firstly, depolarization in the absence of external Ca2+ did not elicit intracellular Ca2+ signals or contractions. Secondly, selective L-type Ca2+ channel blockade (3 μM nifedipine) completely inhibited K+-induced contractions. Thirdly, BAY K8644, an agonist of L-type Ca2+ channels, increased the K+-sensitivity of the contractions. Finally, although intracellular Ca2+ release or Ca2+-induced Ca2+ release through activation of IP3 or ryanodine receptors or Ca2+ re-uptake to the SR have been shown to contribute to K+-induced contractions[2+ release did not account for the biphasic pattern of high K+-induced force and Cai2+ and either phase was solely initiated by L-type Ca2+ influx.As expected, contracractions,37,38, tractions). Hence,2+ channels. L-type Ca2+ currents in isolated SMCs of various tissues and species display a bell-shaped voltage-dependence with maximal currents at 0 to +20 mV[2+ channels starts at −50 to −40 mV with half-maximal activation at −30 mV[+) and complete current inactivation , two populations of L-type Ca2+ channels are expected to contribute to Ca2+ influx and contraction. One population of channels will activate and completely inactivate during the depolarization leading to a transient Ca2+ influx and concomitant contraction , which is near the activation voltage of L-type Ca2+ channels and within the physiological range of VSMCs Vm. As their spiking frequency increased with the amplitude of the depolarization step, fusing to a single fast component at 30 and 50 mM K+ (−36 and −24 mV) similar to the fast component in the step protocol, these events might be related with activation and complete inactivation of L-type Ca2+ channels. Because they occur at physiological Vm of VSMCs, they may have physiological importance. They may be related with the persistent calcium sparklets that are increased in hypertension[The contraction elicited by depolarization of VSMCs has been studied extensively, but haso +20 mV,23,25,40t −30 mV, whereast −30 mV,42. As artension, with ar −50 to − mV, whic+ concentrations studied, a variable population of channels will not completely inactivate and will permit “time-independent” Ca2+ influx via the so-called voltage window[+) should activate a time-independent, non-inactivating Ca2+ influx. Following removal of the fast force component by depolarization in the absence of external Ca2+ and, then, re-adding Ca2+  and a bell-shaped voltage-dependence are paralleled by a tonic force component which increased with [K+ up to 50 or 70 mM (Vm = −20 to −30 mV), but decreased again above 70 mM K+, leading to a bell-shaped [K+-contraction curve. Its voltage range is bounded at negative potentials by channel activation and at more positive potentials by channel inactivation. This agrees with the K + -dependence of the slow force component described in FigureHowever, at all Ke window. Hence,  Figures and6 wem and that changes of Vm within the voltage range of the L-type Ca2+ channel window will stimulate or inhibit Ca2+ influx via L-type Ca2+ channels and the concomitant contraction. Since removal of extracellular Ca2+ led to a decline of intracellular Ca2+ and basal tension in the VSMC of the mouse aorta, a “window” Ca2+ influx appeared to be operative and functional at resting potentials, which are between −40 to −60 mV[+, repolarization) or increase  of external K+ modulates basal tension of the mouse aortic segments, probably via closing and opening of L-type Ca2+ channels because the effects of K+ changes are emphasized by applying BAY K8644  or cromakalim[+ might pull Vm out of the window, thereby decreasing L-type Ca2+ influx, inducing vasodilatation, elevated arterial compliance[K of −85 mV at 5.9 mM K+[2+ and baseline tension, and shifted the [K+-contraction curve to higher K+ concentrations by +6 mM K+ at midpoint.Hyperpolarization of Vith EDHF,36,47-49omakalim, or withmpliance, and hyp−/− mice[m in the L-type Ca2+ channel window leading to increased window L-type Ca2+ influx, basal constriction, decreased arterial compliance, increased myogenic responses and hypertension.On the other hand, it is expected that factors causing depolarization of the membrane potential such as intravascular pressure, hyperte−/− mice, the abs−/− mice might fo2+ channel window along the voltage axis may have profound effects on basal and stimulated Ca2+ influx in VSMC, but also predict that shifts of the activation or inactivation curves of L-type Ca2+ channels affect vasoconstriction and/or dilatation. For example, BAY K8644, which shifts the L-type Ca2+ channel activation curve to hyperpolarized potentials[2+ influx and tone , especially at low K+[m of rat mesenteric arteries from −58 to −82 mV (hyperpolarization to VK)[+-force curve by +5.9 mM K+ at midpoint, Vm at normal K+ of 5.9 mM was calculated to be 19 mV less polarized than VK (−66 mV instead of the Nernstian −85 mV); this is in good agreement with resting Vm of arterial SMCs mentioned in the literature[+, the difference between Vm and VK further diminished from 19 to 7 and 3 mV. Therefore, clamping the segments with K+ was, in our hands, a good technique to restrain the resting Vm of the SMCs.Voltage-clamp of multicellular aortic segments with electrophysiological techniques is impossible with current methods because of temporal and spatial voltage heterogeneity. Therefore, we clamped the aortic rings with extracellular Kat low K+ (seeAddn to VK) and thatterature,47 physiology of conduit blood vessels. Hence, any modulation of L-type Ca2+ influx in VSMC is expected to affect endothelium-dependent and -independent Ca2+ mobilization and related vasomotor responses of blood vessels or arterial compliance. Window L-type Ca2+ influx may underlie the reduced relaxing efficacy of NO in mouse aorta when the contraction is elicited mainly via L-type Ca2+ influx[m of the VSMC or the expression/properties of the population of L-type Ca2+ channels, favoring one or another isoform, might have implications for the window Ca2+ current, influx and contraction, for the sensitivity to L-type Ca2+ channel blockers and NO, for the arterial compliance and for the effects of hypertension on the cardiovascular system.Besides a phasic, fast transient Ca+ influx. Therefo-1, i.p.). The thoracic aorta was carefully removed, stripped of adherent tissue and dissected systematically. Starting at the diaphragm, the ascending thoracic aorta was cut in segments of 2 mm width (5 to 6 segments). Vessels were immersed in Krebs Ringer solution  with (in mM): NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, CaEDTA 0.025, and glucose 11.1. When Ca2+ was omitted from the KR, 1 mM EGTA was added (further named 0Ca) and, hence, to restore 2.5 mM free Ca2+, 3.5 mM Ca2+ was added to 0Ca (further named 0–3.5Ca) from a 1.75 M CaCl2 stock. High K+- solutions were prepared by replacing NaCl with equimolar KCl.The studies were approved by the Ethical Committee of the University of Antwerp, and the investigations conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . C57Bl6 mice  were used at the age of 4 to 7 months. Animals were euthanized under pentobarbital anesthesia  inverted (inside out) endothelium-denuded segments were mounted in the wire myograph, incubated with HEPES-buffered bathing solution  and impaled with glass intracellular microelectrodes (filled with 2 mM KCl and tip resistances between 65 and 90 MΩ). Vm was measured with a HEKA EPC9 amplifier  in the zero current clamp mode and recorded on paper (Gould pen writer). Only measurements of Vm starting with a sharp decrease of Vm upon impalement and a sharp return to approximately 0 mV upon withdrawal of the electrode were considered.To measure resting membrane potentials . The holding potential from which voltage steps would be applied was mimicked by holding the segments at each K+ concentration before a subsequent challenge with higher K+ (cumulative depolarization protocol).To simulate voltage clamp protocols used in electrophysiological studies, extracellular K+. Isometric force was reported in mN. Nitric oxide (NO) formation was inhibited with a combination of 300 μM NΩ-nitro-L-arginine methyl ester (L-NAME) and 300 μM NΩ-nitro-L-arginine (L-NNA) and to avoid any vasomotor interference due to prostanoids, 10 μM indomethacin was present.Aortic segments were mounted in 10 ml organ baths, tension (mN) was measured isometrically with a Statham UC2 force transducer (Gould) connected to a data acquisition system  as described[2+ signals. Segments were loaded for 120 minutes with aerated  KR containing 10 μM Fura-2 AM, 1 mg/ml bovine serum albumin and 0.02% Pluronic at room temperature. Then, temperature was raised to 37°C and the segment was set to its normalized diameter[i2+  after subtraction of background emission values, which were determined by adding 2 mM MnCl2 at the end of each experiment. Contractile force was measured simultaneously and reported in mN mm-1[Segments were mounted in a wire (40 μm) myograph above an inverted microscope  after removal of the endothelium by rubbing their interior with a braided silk wax to avoid interference by endothelial Cadiameter. The sinn mN mm-1.max) and the negative logarithm of the concentration resulting in 50% of the maximal effect (pEC50) for each vessel segment. Two-way ANOVA with Bonferroni post-test (concentration-response curves) and paired or unpaired t-test  were used to compare means of the different experimental groups. A 5% level of significance was selected.All results are expressed as mean ± sem; n represents the number of mice. Time-force curves were fitted with a bi-exponential function revealing amplitudes and time constants of first (fast) and second (slow) components. Concentration-response curves were fitted with sigmoidal concentration-response equations with variable slope, which revealed maximal responses  was obtained from Sanofi , indomethacin from CERTA (Belgium), L-NNA, L-NAME, nifedipine, ryanodine, 2-APB, HA-1077 dihydrochloride from Sigma , Fura 2-AM from Molecular Probes , (±) BAY K8644, levcromakalim, glibenclamide from TOCRIS , Y-27632 dihydrochloride from Abcam Biochemicals .2+: Calcium; VSMC: Vascular smooth muscle cell; K+: Potassium; 2-APB: 2-aminoethoxydiphenyl borate; L-NAME: NΩ-nitro-L-arginine methyl ester; L-NNA: NΩ-nitro-L-arginine; NO: Nitric oxide; SERCA: Sarco-endoplasmic reticulum calcium ATPase; SR: Sarcoplasmic reticulum; eNOS: Endothelial nitric oxide synthase; VK: Equilibrium potential for K+ ions; Vm: Membrane potential.CaThe authors declare that they have no competing interests.All authors read and approved the final manuscript. PF and CVH conceived of the study, designed the experiments, collected and analyzed the data; PF and HB drafted the manuscript, PF, CVH, JVL, DS, WM, GDM and HB participated in interpretation of the results and final draft of the manuscript.Additional information.Click here for file


During the first meiotic prophase, programmed DNA double-strand breaks (DSBs) are distributed non randomly at hotspots along chromosomes, to initiate recombination. In all organisms, more DSBs are formed than crossovers (CO), the repair product that creates a physical link between homologs and allows their correct segregation. It is not known whether all DSB hotspots are also CO hotspots or if the CO/DSB ratio varies with the chromosomal location. Here, we investigated the variations in the CO/DSB ratio by mapping genome-wide the binding sites of the Zip3 protein during budding yeast meiosis. We show that Zip3 associates with DSB sites that are engaged in repair by CO, and Zip3 enrichment at DSBs reflects the DSB tendency to be repaired by CO. Moreover, the relative amount of Zip3 per DSB varies with the chromosomal location, and specific chromosomal features are associated with high or low Zip3 per DSB. This work shows that DSB hotspots are not necessarily CO hotspots and suggests that different categories of DSB sites may fulfill different functions. For sexual reproduction, meiosis is an essential step ensuring the formation of haploid gametes from diploid precursors of the germline. This reduction in the genome's content is achieved through a specialized type of division, where a single round of DNA replication is followed by two successive rounds of chromosome segregation. The first round separates the homologs. For this to faithfully occur, homologous chromosomes pair with each other and experience recombination, catalyzed by the formation of programmed double-strand breaks (DSBs). Upon their repair, a subset of DSBs will generate crossovers, which result from an intermediate that creates a physical link between homologs and allows their correct segregation by the meiotic spindle. DSBs, as well as crossovers, do not occur randomly along chromosomes but at preferential places called hotspots. To ask if all DSB hotspots also give rise to high crossover frequency, we have systematically compared the map of DSBs with that of a protein, Zip3, which we show preferentially binds to DSB sites that are being repaired with a crossover. We discovered that several DSB hotspots rarely produce crossovers, meaning that the decision to repair a DSB with a crossover can be influenced by specific chromosomal features. During meiosis, the programmed formation of DNA double-strand breaks (DSBs) and their repair by homologous recombination ensures that crossovers (CO) occur between homologous chromosomes. COs promote the accurate segregation of homologs at the first meiotic division, thus avoiding aneuploidy, which is a common cause of birth defects and congenital diseases. In all species, two to 30 times more DSBs are formed than COs, indicating that only a subset of all DSBs formed in a cell are repaired through a pathway that will give rise to a CO. The remaining DSBs are repaired by other homologous recombination pathways, such as the synthesis dependent strand annealing (SDSA) mechanism, symmetrical Holliday junction resolution or Holliday junction dissolution Schizosaccharomyces pombe, strong discrepancies were found between the DSB map and the CO frequencies DSBs and COs tend to occur more frequently at preferred sites, or hotspots. It is not known whether DSB hotspots are also CO hotspots, or whether DSB repair is modulated by DSB localization on a chromosome. This question could be answered by comparing a high resolution genome-wide map of CO frequencies to the existing high resolution maps of DSBs, for instance in budding yeast e.g., . Neverthin vitro, such as the Mer3 helicase, which promotes D loop extension and the Msh4–5 heterodimer, which stabilizes dHJs. This group also includes Zip1, the central element of the synaptonemal complex (SC), as well as Zip2, Zip3, Zip4 and Spo16 that might promote SC formation through Zip1 polymerization between homolog axes C. elegans (ZHP-3) and in mammals (RNF212) and is considered to be a SUMO E3 ligase that sumoylates chromosome axis proteins, thus promoting SC polymerization. Indeed, the Zip3 sequence includes a SUMO Interacting Motif (SIM) and a C3H2C3 Ring-Finger Motif (RFM) that are important for Zip3 in vitro E3 ligase activity and necessary for SC polymerization and correct sporulation Several factors affect CO formation and their sites of action may reflect how a DSB is repaired. A group of proteins collectively termed “ZMM” is necessary for the formation of about 85% of all COs in budding yeast spo11 mutant strains in which the number of DSBs but not of COs is reduced (a phenomenon known as CO homeostasis), the number of Zip3 foci follows the CO variation Indirect evidence suggests that ZMMs localize at CO-designated sites, but this has never been demonstrated. ZMMs form foci during meiotic prophase at the time of recombination We thus mapped Zip3 binding sites along individual genomic regions and genome-wide during budding yeast meiosis and then determined the features that influence its distribution. We show that Zip3 association with chromosomes is dynamic, occurring first with centromeres, in a DSB-independent manner, then with meiotic chromosome axes upon DSB formation and finally with DSB sites upon joint molecule formation, the preferred intermediate for CO production. These features establish Zip3 as a marker of CO-designated sites. Genome-wide mapping of Zip3 recruitment to DSB sites demonstrates the existence of different types of DSB hotspots based on CO production.BUD23 promoter hotspot on chromosome 3 form and reach a maximum at 3–4 hr, before getting repaired  and qPCR and yeast strains in which Zip3 was C-terminally tagged at its endogenous locus with three copies of the Flag epitope. Strains expressing the ZIP3-His6-FLAG3 allele showed normal meiotic progression and spore viability , showing that the tagged protein is functional. During a meiotic time-course, DSBs monitored at the repaired . Zip3 shrepaired . At thisrepaired , indicatzip3I96K mutant) or the RFM (zip3H80A mutant) motif. Both mutated proteins were timely induced during meiosis, but they lacked the characteristic lower migrating bands that correspond to sumoylated Zip3 zip3 mutants (zip3 null mutants (data not shown). These findings indicate that Zip3 SUMO binding and E3 ligase activities are essential for Zip3 association with chromosomes and all its functions in meiosis. SUMO binding could be directly involved in Zip3 recruitment to all these chromosome locations or indirectly, if required only for the initial Zip3 enrichment at centromeres, and if this is an essential step for the subsequent recruitment of Zip3 to axes and DSB sites.As Zip3 might be a SUMO E3 ligase, we investigated whether interaction with SUMO regulated Zip3 binding to the different chromosomal structures. To this aim, we mutated the Zip3 SIM , at the three sites examined  intermediate, a crossover-specific intermediate, but are strongly impaired in the following step, second end capture, which leads to double Holliday junction formation and crossover resolution rad52Δ mutants, we detected centromere and axis association delayed but to nearly wild-type levels, but a strongly reduced binding of Zip3 to the three DSB sites . This led to a decrease of the low migrating forms of Zip3 due to phosphorylation  and kinetics of DSB formation and repair were as in wild-type , in which the alternative CO pathway is inactivated zip3-4AQ mus81Δ mutants by physical analysis of the EST3-FAA3 DSB site with flanking markers. In our hands and at the hotspot examined, mutation of MUS81 did not affect CO formation in both strains, and CO was even slightly stronger in each case compared to its MUS81 counterpart  and three sites with relatively lower Zip3 accumulation   . The intF1-ADH3)  and we tF1-ADH3) . The CO/DSB site .set1Δ strain, in which DSBs are reduced and redistributed to new sites PES4, a strong set1Δ DSB site, was highly enriched in Zip3, whereas ARG3, another strong set1Δ DSB site, was not , we identified “low-Zip3” DSB sites (n = 166 sites) and “high-Zip3” DSB sites (n = 142 sites)  or low (113 sites), based on the peak signal intensity like we did for the Zip3 peaks, we found that the Zip3 signal was significantly lower in low-rad50S DSBs . This further strengthens our observation that at least a subset of low-Zip3 DSB sites also shows reduced DSB formation in the rad50S mutant, suggesting that they have distinct properties.When we classified the DSBs in the 50S DSBs . Overallrad50S DSB sites , but the low-Zip3 DSBs were significantly further away from a set1Δ Rec8 peak or a Red1 peak than the high-Zip3 DSB sites . The protein responsible for Zip3 loading onto axis sites could be an axis protein that is phosphorylated by the Tel1/Mec1 kinases, such as Hop1 zip1Δ mutant. It was proposed that at centromeres, Zip1 stabilizes Smt3 chains, made by other SUMO ligases acting in early meiosis, thus favoring Zip3 binding to centromeres. Our data confirm previous cytological observations zip3 SUMO ligase and the zip1Δ mutants are consistent with a previously proposed model zip1Δ mutant, Zip3 association with DSB sites was strongly decreased.We observed a reduced recruitment of Zip3 to all chromosomal regions in the ndt80Δ mutant than in the wild-type. The ndt80Δ mutant accumulates non-cleaved dHJs and thus our data are consistent with the proposed role of Zip3 and the ZMM in general to stabilize the crossover-designated intermediates from D-loop dismantling and later from dHJ dissolution by activities exerted by anti-crossover factors such as Sgs1 ndt80Δ cells. This may be due to a change of structure within the synaptonemal complex that persists in this mutant and that alters the association of sites undergoing dHJ with axis-associated sites, and renders these closer to strong DSB sites and thus more closely associated with Zip3. It would be interesting to determine if this increase of Zip3 association is seen for all axis-associated sites or only those that are close to strong DSB sites.Interestingly, Zip3 foci persisted more on DSB sites in the We detected a negative influence of the centromere on the relative binding of Zip3 to DSB sites. However, Zip3 binding was not abolished, although these regions show few CO and NCO events and have been suggested to repair their DSBs mostly using the sister chromatid rad50S mutant . For calculation of the ratio between CO and DSB per cell, we divided the % of cells that received a CO (genetic distance * 2) by the % of cells that received a DSB .For genetic distances on chromosomes III, VII and VIII, haploids were mated at 30°C on YPD supplemented with 1% Adenine for 5 hr before replica-plating on solid sporulation medium (1% potassium acetate) and incubated at 30°C for at least 48 hr. For recombination between hemizygous drug resistance markers, diploids were grown on YPD plates and then replica plated on sporulation medium at 30°C for at least 48 hr. Asci were dissected on YPD supplemented with 1% Adenine and replica-printed to the appropriate media to check for marker status. P , NPD  and T (tetratype) were scored to calculate the genetic distances as described in Genomic DNA was prepared, analyzed and DSB or CO frequency was determined as described BUD23 on chromosome 3; DSB2 is in the promoter of ECM3 on chromosome 15 and DSB3 in the promoter of RIM15 on chromosome 6. Axis site was chosen from the Rec8 binding data of CDC39, on chromosome 3. Primer positions are in For each time-point, cells were processed and ChIP was performed as described set1Δ Zip3-Flag 6 and 7 hr ChIP-chip assays, a very high signal was obtained, and we adjusted the threshold to 5 to obtain a number of Zip3 peaks comparable to that of the other experiments. High Zip3 DSB sites were DSB sites that coincide with a Zip3 peak the signal intensity of which differed by less than 50 ranks from that of the DSB site; Low Zip3 DSB sites were DSB sites either not bound by Zip3 or that coincide with a Zip3 peak the signal intensity of which was at least 100 ranks lower than that of the DSB site.Immunoprecipitated DNA and whole-cell DNA were amplified, labeled and hybridized to Agilent 44 k yeast whole genome oligonucleotide arrays as described http://downloads.yeastgenome.org/curation/chromosomal_feature/) of the last update from July of 2010.For the chromosome coordinates, we used the Saccharomyces Genome Database features  ±1.5 times the interquartile range (whiskers). p value indicates the result of a Wilcoxon test between the two indicated time-points.Genome-wide ChIPchip analysis of Zip3 at 3, 4 and 5 h in meiosis. (A) qPCR analysis of the Zip3-Flag ChIP samples used for ChIP-chip analysis. Zip3 association was monitored at the indicated regions in a wild-type strain (ORD9670). The average values from two independent time-courses are shown. The three red arrows indicate the time-points that were used in our ChIPchip analysis. (B) Global temporal variation of Zip3 association with centromeres, axis-association sites and DSBs. For each category, the following regions were considered: centromeres , Rec8, Red1 and DSBs . The decile-normalized ratios after denoising and smoothing using a 2 kb window are indicated. Boxplots show the median (line), 25(TIF)Click here for additional data file.Figure S2Genome-wide profiles of Zip3 localization. Average ChIP-chip Zip3-Flag decile-normalized ratios from two independent wild-type (ORD9670) meiotic time-courses are plotted after denoising and smoothing with a 1 kb window along the 16 chromosomes. Black circles indicate the centromere. Same experiment as in (TIF)Click here for additional data file.Figure S3spo11Δ mutant and Rec8 Flag. Average decile-normalized ratios are plotted along the 16 chromosomes after denoising and 1 kb window smoothing. Green circles indicate the centromere. Rec8 data are from spo11Δ at 3 hr is from ORD9684 strain.Genome-wide profiles of Zip3 ChIP at 3 hr in meiosis, Zip3 in a (TIF)Click here for additional data file.Figure S4dmc1Δ mutant (raw data from Genome-wide profiles of Zip3 ChIP at 4 hr and ssDNA accumulated at DSB ends in a (TIF)Click here for additional data file.Figure S5zip3-6AP mutant strain (VBD1093) during a meiotic time-course. Zip3-Flag was monitored by western blotting with an anti-Flag antibody. Pgk1 served as loading control. (B) Meiotic progression in the same time-courses as in (A). Nuclear divisions were monitored by DAPI staining. (C) Monitoring of Zip3 binding in the same time-courses as in (A) and (B) by ChIP with an anti-Flag antibody and revealed by qPCR using primer pairs that cover the indicated regions.Mutation of the Zip3 consensus phosphorylation sites for the CDK kinase has no effect on Zip3 association with DSB sites. (A) Zip3-Flag expression in a wild-type (ORD9670) and (TIF)Click here for additional data file.Figure S6SPO11 ZIP3: ORD9670 (205 tetrads); spo11YF/HA ZIP3: VBD1191 (124 tetrads); spo11YF/HA zip3-4AQ: VBD1192 (134 tetrads).Spore viability in strains with reduced DSB formation and wild-type Zip3-Flag or mutant Zip34AQ-Flag. The proportion of 4, 3, 2, 1 or 0 viable spore per tetrad is indicated for each strain. (TIF)Click here for additional data file.Figure S7ChIP-chip profiles for Rec8, ssDNA and Zip3 around high- and low-Zip3 DSB sites. The actual site is at the center of each plot×axis. Decile-normalized ratios are represented, after denoising and smoothing with a 2 kb window. Dots indicate sites were a peak was detected. Same strains and experiments as in (TIF)Click here for additional data file.Figure S8Schematic representation of the hemizygous flanking marker configuration used to assess genetic distances.(TIF)Click here for additional data file.Figure S9dmc1Δ cells and analyzed by Southern blotting. The brackets on the side of each panel indicate the physical interval comprised between the genetic recombination markers used to measure genetic distances. Red arrows indicate new DSB due to the insertion of a flanking marker. Below each panel is indicated the DSB frequency measured from at least two independent time-courses ± standard deviation. EST3-FAA3: with flanking markers: strain VBD1168; no markers: VBD1172. ATG2-LAP3: with flanking markers: strain VBD1218; no markers: VBD1172. COG7-LEU1: with flanking markers: strain VBD1172; no flanking markers: VBD1168. ISF1-ADH3: with flanking markers: strain VBD1170; no flanking markers: VBD1172.DSB frequencies in the chosen high-Zip3 and low-Zip3 intervals in the absence or presence of hemizygous flanking markers. Genomic DNA was extracted at the indicated time during meiosis from (TIF)Click here for additional data file.Figure S10set1Δ with varying frequencies. (A) ChIP monitoring of Zip3-Flag association with the indicated regions during a meiotic time-course in set1Δ cells (VBD1005). (B) Comparison of the profiles between pairs of experiments. The name of each experiment is indicated, as well as the number of peaks in common between the two experiments and as percentage of the peaks of the first experiment. Pcorr assesses the linear Pearson's correlation coefficient between the profiles of the two experiments after denoising and smoothing with a 2 kb window. set1Δ DSB: raw data are from set1Δ Rec8: data are from dmc1Δ DSBs in set1Δ strains in the PES4 and ARG3 regions, two sites with increased DSB frequency in the set1Δ mutant. set1Δ Zip3-Flag data are from the same time-course as in (A). set1Δ DSB raw data are from the Rpa ChIP-chip at 7 hr in a set1Δ dmc1Δ strain dmc1Δ strains at 7 hr in meiosis (ORD9624) and values are from eight (PES4) and six (ARG3) independent experiments. Genetic distances were determined by scoring the segregation of hemizygous resistance markers flanking each interval (see set1Δ DSB frequencies in the dmc1Δ (ORD9624) and rad50S (VBD1117) backgrounds at PES4 and ARG3 sites. DSB formation was measured by Southern blotting as described in set1Δ DSBs based on the presence of an associated Zip3 peak at 6 hr the signal intensity of which was less than 50 ranks away from that of the DSB site. Low-Zip3 DSB sites (n = 39) were selected among the strongest 200 set1Δ DSBs based on the absence of a Zip3 associated site, or on the presence of a Zip3 site with signal intensity at least 150 ranks below that of the DSB site. The set1Δ Zip3 at 6 hr, 7 hr, dmc1Δ DSB and Rec8 data sets are the same as in (B). Red1 binding raw data are from Zip3 associates with DSB hotspots in rval see . (D) Com(TIF)Click here for additional data file.Figure S11set1Δ ChIP-chip of Zip3 at 6 hr and RPA accumulated at DSB ends in a set1Δ dmc1Δ mutant (raw data from Genome-wide profiles of (TIF)Click here for additional data file.Figure S12Association of Zip3 with centromere-proximal DSBs. Examples of Zip3 and DSB signals at four centromere regions and one chromosome arm. Graphs represent decile-normalized data after denoising and smoothing with a 2 kb window of Zip3 ChIP-chip at 4 hr or ssDNA at DSB. Same data as in (TIF)Click here for additional data file.Figure S13rad50S DSBs (see details in the text). The rad50S and dmc1Δ DSB datasets are from Features of the low-Zip3 DSB sites that are not low-(TIF)Click here for additional data file.Protocol S1Contains details about yeast strains construction, enzymes and probes used for DSB mapping and position of qPCR primers.(DOC)Click here for additional data file.Table S1zip3-4AQ mutation on genetic distances in intervals along three chromosomes. Map distances and standard errors  were calculated from parental ditypes (PD), non-parental ditypes (NPD) and tetratypes (T) as described in the P values are for G tests performed on parental ditype, non-parental ditype, and tetratype segregation patterns for pairwise comparison between wild-type ZIP3-Flag and the zip3-4AQ-Flag mutant. Same strains as in Effects of the (XLSX)Click here for additional data file.Table S2Genetic distances at High- and Low-Zip3 DSB sites.(XLSX)Click here for additional data file.Table S3spo11Δ (ORD9684) or set1Δ (VBD1005) strains and for untagged control (ORD7339). Average decile-normalized ratios calculated from two independent experiments for each condition are included, as well as denoised ratios after smoothing with a 2 kb window. The RefNumber column shows the reference provided by the manufacturer (Agilent) to allow easy alignment with characteristic features or other data using the same microarray platform. The raw and processed data have been deposited in the Genome Omnibus Database (GSE40563).Processed ChIP-chip data for Zip3-Flag in wild-type (ORD9670), (TXT)Click here for additional data file.Table S4Strains used in this study.(DOC)Click here for additional data file.


Resting oxygen consumption of isolated muscles  measured polarographically at 30°C was ~20% higher (P<0.05) in soleus (0.326 ± 0.022) than in EDL (0.261 ± 0.020). In order to quantify the specific contribution of Ca2+ cycling to resting metabolic rate, the concentration of MgCl2 in the bath was increased to 10 mM to block Ca2+ release through the ryanodine receptor, thus eliminating a major source of Ca2+ leak from the sarcoplasmic reticulum (SR), and thereby indirectly inhibiting the activity of the sarco(endo) plasmic reticulum Ca2+-ATPases (SERCAs). The relative (%) reduction in muscle VO2 in response to 10 mM MgCl2 was similar between soleus (48.0±3.7) and EDL (42.4±3.2). Using a different approach, we attempted to directly inhibit SERCA ATPase activity in stretched EDL and soleus muscles (1.42x optimum length) using the specific SERCA inhibitor cyclopiazonic acid , but were unsuccessful in removing the energetic cost of Ca2+ cycling in resting isolated muscles. The results of the MgCl2 experiments indicate that ATP consumption by SERCAs is responsible for 40–50% of resting metabolic rate in both mouse fast- and slow-twitch muscles at 30°C, or 12–15% of whole body resting VO2. Thus, SERCA pumps in skeletal muscle could represent an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity.The main purpose of this study was to directly quantify the relative contribution of Ca Under basal conditions in skeletal muscle, sarco(endo) plasmic reticulum Ca2+-ATPases (SERCAs) are responsible for maintaining a >104-fold Ca2+ concentration gradient across the sarcoplasmic reticulum (SR) membrane and for keeping the cytosolic free Ca2+ concentration ([Ca2+]f) at or below 100 nM f which activates other cytosolic Ca2+-dependent ATPases, most notably the acto-myosin ATPase. Chinet et al. [2+ uptake from the resulting rise in [Ca2+]f and development of contracture on muscle energy expenditure. In order to accurately quantify the contribution of Ca2+ cycling to resting metabolic rate in skeletal muscle, SERCA activity must be inhibited directly in conjunction with myosin ATPase activity. This can be accomplished by employing stretch to eliminate filament overlap prior to inhibiting SERCA activity i would be highly inhibitory to myosin ATPase activity due to a lack of thin filament activation [o) to prevent thick and thin filament overlap to determine the metabolic cost of crossbridge cycling in resting EDL and soleus muscles.Since elevated [Mg]tivation , a subse2 was recorded at 1.42 Lo in the presence of cyclopiazonic acid (CPA), a highly specific and potent SERCA inhibitor ) and the amounts of SERCA1a or SERCA2a in a given sample  were determined from standard curves on the same Western blot i indicated by the indo-1 fluorescence ratio . The average resting VO2 (µL/g wet weight/s) at 30°C was ~20% higher (P<0.05) in soleus (0.326 ± 0.02) compared with EDL (0.261 ± 0.02). Pre-stretching the muscles had no effect (P>0.05) on resting VO2 of either soleus or EDL muscles, with stretched VO2 equal to 104 ± 9 and 102 ± 10% of resting VO2 at Lo, respectively  of incubation were not different (P>0.05) than measurements taken from 60–90 min (Washout) of incubation  and soleus (48.0±3.7) muscles .The O2 tracings of leak (No Muscle), rest, and 160 µM CPA trials for a soleus muscle are depicted in 2 . Importantly, there was no increase in resting tension following the addition of CPA in the stretched muscle indicating that myosin ATPase was not activated during the addition  were compared in EDL and soleus by quantitative Western blotting as described under “Materials and Methods”  of MgCl2 and thus indirectly inhibited SERCA activity, we showed that the relative (%) contribution of Ca2+ dependent ATP turnover to resting metabolic rate is substantial (i.e. ~40-50%) and not different between EDL and soleus.We hypothesized that previous approaches ,10 usingfindings ,10, when2 could be due to either activation of non-myosin, Ca2+-dependent ATP consuming processes, or incomplete inactivation of SERCA. CPA is known to be cell permeable and the concentrations used in this study exceeded those known to maximally inhibit SERCA activity in skinned fiber preparations i. However, high extracellular Mg2+ has been shown to change the surface charge potential and shift the contraction threshold of frog skeletal muscle fibers to less negative potentials, mimicking hyperpolarization f is constant in resting skeletal muscle, basically 2 main factors determine the ATP turnover rate by SERCAs in resting muscle: 1) the rate of Ca2+ leakage out of the SR and 2) the stoichiometry of Ca2+ uptake by SERCAs (i.e. Ca2+/ATP ratio). Measurements in mechanically skinned fibres from rat EDL and soleus indicate that under normal resting conditions, Ca2+ leaks out of the SR at approximately the same absolute rate in both muscles  in the SR at sufficiently low levels to prevent high rates of Ca2+ leakage through the high density of SERCA pumps. Assuming that rat and mouse fibres are comparable and the SR Ca2+ leak rates are the same in mouse EDL and soleus, the similar rates of ATP turnover by SERCAs we observed in EDL and soleus would therefore suggest that the efficiency of Ca2+ uptake by SERCAs in soleus and EDL is also similar, at least under resting conditions.In this study, we measured rates of oxygen consumption at 30er study , our fin studies ,13,34. T muscles ,36. CRCss regard ,36; thern soleus ,38 and tn soleus ,40. In t2+ transport to ATP hydrolysis by SERCAs is considered to be optimal as it corresponds to the stoichiometry of two Ca2+ binding sites and one ATP binding site on each SERCA pump  concentration in the fibers (~0.1 µM vs. ~10 µM) were higher in the present study. Higher coupling ratios have been reported for rabbit skeletal muscle preparations and, in contrast with our calculations, the Ca2+/ATP ratio was higher in slow-twitch muscle SR vesicles (i.e. 0.98 Ca2+/1ATP) compared with fast-twitch skeletal muscle SR vesicles (i.e. 0.37 Ca2+/1ATP) [A 2:1 ratio of CaRCA pump ,42. Howe the rat ,36,43. T Cellini ,36,43 fo muscles –46, we areatment  and [ADPreatment . Comparereatment ,36,43, b2+/1ATP) . The cou2+/1ATP) . In cont2+/1ATP) .2 or the relative contribution of SERCA activity to the metabolic rate of the different fiber types. It is worth noting that Murphy et al. [In adult C57BL/6 mice, the EDL contains >90% type II fibers (mostly IIB) and <10% type I fibers whereas the soleus contains ~50% type I and type II fibers (mostly IIA) ,50. Our y et al.  reportedy et al. . Importa2. It is also well known that SERCAs contribute ~30-40% to the energy cost associated with muscle contraction (for review, see It is well known that SERCAs play an important role in thermogenesis ,47 but S°C. These results have important implications for the basic understanding of muscle energetics and suggest that the SERCA pump in skeletal muscle represents an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity and other metabolic disorders.In summary, the results from this study support the conclusion that ATP consumption by SERCAs is responsible for 40-50% of resting metabolic rate in both mouse EDL and soleus muscles at 30