Ciliopathies are a group of genetic disorders caused by a failure to form functional cilia. Due to a lack of structural information, it is currently poorly understood how ciliopathic mutations affect protein functionality to give rise to the underlying disease. Using X-ray crystallography, we show that the ciliopathy-associated centriolar protein CEP120 contains three C2 domains. The point mutations V194A and A199P, which cause Joubert syndrome (JS) and Jeune asphyxiating thoracic dystrophy (JATD), respectively, both reduce the thermostability of the second C2 domain by targeting residues that point toward its hydrophobic core. Genome-engineered cells homozygous for these mutations have largely normal centriole numbers but show reduced CEP120 levels, compromised recruitment of distal centriole markers, and deficient cilia formation. Our results provide insight into the disease mechanism of two ciliopathic mutations in CEP120, identify putative binding partners of CEP120 C2B, and suggest a complex genotype-phenotype relation of the CEP120 ciliopathy alleles.  •The centriolar protein CEP120 contains three adjacent C2 domains (C2A–C2C)•in vitroTwo ciliopathy-associated mutations in CEP120 C2B destabilize this domain •In cells, both mutations reduce cellular and centrosomal CEP120 levels•Both mutations impede distal centriole function and block cilia formation Joseph et al. structurally and functionally characterize a conserved C2 domain of the centriolar protein CEP120, which harbors two distinct ciliopathy-associated mutations. Their results suggest that, by destabilizing the C2 domain, these mutations reduce cellular and centrosomal CEP120 levels, thereby causing abnormal distal centriolar function and defective ciliogenesis. Cilia are hair-like protrusions of the plasma membrane. They are essential cellular organelles with multiple functions, such as cell motility and liquid movement, and also play a crucial role in several major signaling pathways. Cilia are formed when centrioles  dock against the cell membrane and extend their peripheral microtubule array to form the ciliary axoneme. Centrioles display a defined polarity, with microtubule minus and plus ends marking the proximal and distal tips, respectively. At the start of ciliogenesis, the mother centriole is converted into a basal body via recruitment of a Golgi-derived ciliary vesicle onto its distal end. This process, which is a prerequisite for basal body docking, is facilitated by components of the centriole distal appendages, including CEP164 . Distal Ciliopathies are a group of human genetic diseases in which cilia formation or function is impaired. So far, multiple mutations in over 50 genes  have beeOur understanding of how ciliopathies arise from a given mutation and how mutations relate to the resulting ciliopathies is currently limited due to several factors. First, structural information on most of the underlying proteins is lacking, limiting our grasp of how these mutations affect protein structure and function. Second, the cellular mechanisms by which a given ciliopathic mutation translates into a disease are often incompletely understood. Third, cilia are complex cell organelles with multiple functions and a proteome of up to 1,000 proteins , complicCEP164, TALPID3, CSPP1, PLK4, and CEP120, also cause ciliopathies, raising the question of how their dysfunction mechanistically translates to the corresponding disease  of CEP120. Oreochromis niloticus (O.n.) (but not from Homo sapiens [H.s.]) we obtained diffracting crystals that allowed a structure determination at a resolution of 1.6 Å (Danio rerio and C2C from Mus musculus) to a resolution of 1.4 Å and 1.9 Å, respectively  adopt the PLC δ1-like topology II and are structurally similar to each other , with major differences found mainly in their loop length. Analysis of CEP120 homologs across different organisms showed that most metazoan CEP120 proteins possess an organization with three C2 domains that are followed in sequence by a coiled-coil region A. While Oreochromis niloticus, the equivalent residues (V195 and A200) point inward toward its hydrophobic core , we obtained diffraction-quality crystals that allowed a successful structure determination to a resolution of 2.1 Å,  mutant structure revealed an overall preservation of the C2 domain fold and only subtle structural changes. In the WT structure, the O.n. A200 residue is located at the end of β strand 1 and its side chain points inward toward the hydrophobic interior of the domain. The replacement of this alanine by proline causes a change in the main-chain dihedral angles of the preceding residues, resulting in a local structural change (O.n.-Phe199 (H.s.-Phe198) makes a main-chain/main-chain hydrogen bond to O.n.-Gly333 (H.s.-Gly333) that cannot be maintained in the A199P mutant due to the conformational constraints imposed by the adjacent proline residue. In the mutant structure, the loss of this hydrogen bond is compensated by a peptide bond flip, allowing the main-chain carbonyl O of O.n.-Ala198 (H.s.-Ala197) to form a new hydrogen bond with O.n.-Gly333 (H.s.-Gly333). Both A198 and F199 are involved in forming a beta-bulge region in β strand 1, facilitating this flip. Residues in close contact to the O.n.-A200P (H.s.-A199) side chain are also affected by the mutation. The most significant conformational change is observed for O.n.-F244 (H.s.-F243), the side chain of which moves to avoid a steric clash with the bulky pyrrolidine ring of the proline. Other structural differences between the WT and A200P mutant, in particular the conformational change in loop 1 and loop 3, are most likely caused by different crystal-packing interactions.The CEP120 C2B  and V195 (H.s.-V194), are essentially invariant in both species with the exception of human I196, I285 and V332 that in O.n. are substituted by V, V, and L, respectively.The WT and mutant C2B structures were derived from gy model  suggest O.n. C2B A200P (H.s. A199P) mutant are relevant for the human homolog in solution, we turned to nuclear magnetic resonance (NMR) spectroscopy, which enabled us to study WT and both V194A and A199P mutant H.s. CEP120 C2B under the same conditions. Backbone resonances of the 13C, 15N double-labeled WT H.s. CEP120 C2B were assigned at 30°C to increase the sensitivity of triple-resonance experiments  spectra of WT and V194A as well as A199P mutant H.s. CEP120 C2B at 20°C using 15N-labeled samples. At this temperature, WT and mutant samples were stable over the time course of the experiments. An overlay of WT and mutant spectra , which measures global excess heat absorption during that process A. Striki1H, 15N BEST-TROSY spectra of 15N labeled WT, V194A and A199P H.s. CEP120 C2B were acquired at 20°C, 35°C, and 37°C  or the JATD (A199P) mutation into the CEP120 gene by clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein (Cas9) (CRISPR-Cas9) targeting in RPE-1 human retinal pigmented epithelial cells . CHX incubation decreased, but did not abolish, expression of the point mutant proteins despite their much-reduced starting levels (Steady-state levels of CEP120 protein were reduced in both JS (V194A) and JATD (A199P) cells E and S4Fg levels B. Thus, g levels . Using ig levels C. Similag levels  , Shaheen and colleagues  of CEP120 affect residues that point toward its hydrophobic core and lead to its reduced thermostability lleagues  detectedlleagues .Both JS and JATD mutants are strongly defective in cilia formation, possibly due to the aforementioned impaired retention/recruitment of distal-end proteins to the mother centrioles that play major roles in cilia formation . Recent in vitro and ciliogenesis in vivo. These results raise the question of why they provoke diseases with distinct phenotypes. The clinical features of JS and JATD can overlap  was crystallized by the vapor diffusion method with sitting drops (100 nL protein solution + 100 nL reservoir solution) in the JBS 1 screen (JenaBioScience) with a reservoir solution of 100 mM HEPES (pH 7.5), 200 mM MgCl2, and 30% (v/v) PEG-400 at 19°C. Crystals were mounted in reservoir solution after 1 day and frozen in liquid nitrogen.O.n. CEP120165–353 G307S and A200P + G307S (C2B) were crystallized in a similar setup at 19°C with a reservoir solution of 100 mM MES buffer (pH 6.1), 29% (v/v) PEG-400 , or 100 mM MES (pH 6.0), 26% (v/v) PEG-400 , and optionally 2 mM DTT  or with a reservoir solution of 100 mM Tris (pH 8.0) and 30% (w/v) PEG-6000 . Crystals were mounted in reservoir solution after 6 days  or 3 days  or were mounted after 10 days in 100 mM Tris-CL (pH 8.5), 25% (w/v) PEG-6000, and 25% glycerol (A200P + G307S).O.n. CEP120165–353 WT was crystallized at 19°C in a similar setup in 96-well plates containing a linear gradient of reservoir solutions from 30% (v/v) PEG-400, 100 mM MES (pH 6.0) (column 1) to 30% (v/v) PEG-400, 100 mM MES (pH 7.0) (column 12). Crystals were mounted in reservoir solution after 10 days from columns 6–8. All crystals were frozen in liquid nitrogen.M. musculus CEP120436-634 (C2C) was crystallized at 19°C by the vapor diffusion method in sitting drops (1 μL protein solution + 1 μL reservoir solution) with a reservoir solution of 50 mM MES (pH 5.2 [native] or pH 6.1 [SeMet]), 10 mM MgCl2, 200 mM KCl, and 5% (native) or 6% (SeMet) (w/v) PEG-8000. Crystals were mounted in reservoir solution without PEG-8000 containing 30% PEG-400 after 4 days (native) or 5 days (SeMet) and frozen in liquid nitrogen.−/− cells were prepared using a CRISPR-Cas9 method following published protocols  CS2 63 or 100× 1.4 numerical aperture oil-immersion Leica objectives. Images shown throughout the manuscript represent maximum intensity projections of z sections taken every 0.3 μm. Images in Statistical analyses and preparation of graphs were performed using Prism7 (GraphPad Software). Details of the used statistical tests and number of experimental repeats  are reported for each dataset in the figures and figure legends.


Fast Ito channels can be produced from the Shal family KCNDE gene product Kv4.3s, although accessory subunits including KChIP2.x and DPP6 are also needed to produce a near physiological Ito. In this study, the effect of KChIP2.1 & KChIP2.2 , alone or in conjunction with the accessory subunit DPP6, on both Kv4.3 and hERG were evaluated. A dual Ito and IKr activator, NS3623, has been recently proposed to be beneficial in heart failure and the action of NS3623 on the two channels was also investigated. Whole-cell patch-clamp experiments were performed at 33 ± 1 °C on HEK293 cells expressing Kv4.3 or hERG in the absence or presence of these accessory subunits. Kv4.3 current magnitude was augmented by co-expression with either KChIP2.2 or KChIP2.1 and KChIP2/DPP6 with KChIP2.1 producing a greater effect than KChIP2.2. Adding DPP6 removed the difference in Kv4.3 augmentation between KChIP2.1 and KChIP2.2. The inactivation rate and recovery from inactivation were also altered by KChIP2 isoform co-expression. In contrast, hERG (Kv11.1) current was not altered by co-expression with KChIP2.1, KChIP2.2 or DPP6. NS3623 increased Kv4.3 amplitude to a similar extent with and without accessory subunit co-expression, however KChIP2 isoforms modulated the compound’s effect on inactivation time course. The agonist effect of NS3623 on hERG channels was not affected by KChIP2.1, KChIP2.2 or DPP6 co-expression.Transient outward potassium current (I Recent data suggest that hERG channel current magnitude is influenced by Kv4.3 co-expression to beta subunits. KChIP2 has recently been identified to act as a core transcriptional regulator of cardiac excitability to increases action potential duration Kr, the natural low level of expression of KChIP2 in guinea pig leaves open the possibility of interactions at higher levels of expression. The promiscuous nature of KChIP2 interactions is further underscored by data demonstrating altered L-type Ca current magnitude in murine myocytes lacking KChiP2 and a direct interaction between KChiP2 and Cav1.2 The AP depolarization also activates I+ channels that both increase repolarizing current and prolong post-repolarisation refractoriness have the potential to offer novel antiarrhythmic actions to may restore early repolarization and inhibit dyssynchronous Ca2+ release that occurs consequent to loss of the early repolarization notch of the AP in heart failure to activator, NS5806, has been shown to enhance native ventricular Ito in dog and rabbit and it also increases recombinant Kv4.2 and 4.3 currents to is augmented by NS5806 to a much smaller extent than ventricle, whilst rabbit atrial Ito is paradoxically inhibited by the compound Kr and Ito and to increase repolarization reserve in cellular and multicellular canine preparationsKrXenopus oocytes at 30 µM to (and IKr) in canine preparations (at 1–5 µM) toper se.Small molecule activators of cardiac KThe present study had three aims: first, to compare the gating properties of Kv4.3 co-expressed with either KChIP2.1 or KChIP2.2 with and without DPP6. Second, to examine the effects of NS3623 on Kv4.3 and hERG, in both the presence and absence of KChIP2 and DPP6 in a mammalian cell expression system. Finally, to examine the possibility that KChIP2.1, KChIP2.2 and DPP6 expression may affect Kv11.1 (hERG) by transfecting each β-subunit  in a stably transfected mammalian (HEK 293) cell line which expresses hERG channels.22.1KCND3 gene cloned in pcDNA3, NCBI reference sequence NM_172198.2) KCNIP2 gene cloned in pcDNA3, NCBI reference sequence NM_173195.2) DPP6 gene cloned in pcDNA3.1+, NCBI reference sequence NM_130797.3) and KChIP2.1  were synthesized and sequenced by GenScript .The cDNA constructs coding for human short Kv4.3 isoform 1 precursor . Cells were maintained at 37 °C in a humidity controlled incubator with 5% CO2 atmosphere and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% (v/v) heat inactivated fetal bovine serum , 1% (v/v) non-essential amino acids  and 50 μg/mL Gentamicin . In the case of hERG-expressing HEK 293 cells, the medium was further supplemented with 400 μg/ml G418 selection antibiotic . Prior to transfection, cells were plated for 48 h onto 12-well plates using a non-enzymatic agent  before transfection. Transfection reactions were prepared using OPTI-MEM® I . For Kv4.3 experiments 0.3 μg of DNA was transfected along with either 1 μg of a GFP construct (control) or KChIP2.1/2.2. In experiments where a KChIP2 isoform and human DPP6 were co-transfected, 0.8 μg of each DNA was transfected and compared to a “control” condition, in which 1.6 μg of GFP alone was transfected. For hERG experiments, a matching concentration of GFP DNA was used, in order to have an equivalent DNA concentration across all conditions. Lipofectamine™ 2000 transfection agent  was used at a 2:1 ratio with DNA. The culture medium was replaced 5 h after transfection. After 24 h, cells were collected and re-plated onto 13 mm round glass coverslips, pre-treated for 4 h with 200 μg/mL Poly-D-Lysine . Patch-Clamp recordings started 3 h after plating.HEK 293 cells  transfected with Kv4.3 or a stable HEK 293 cell line expressing wild-type (WT) hERG (Kv11.1) channels (kindly provided by Professor Craig January) 2.32, 2 CaCl2, 10 HEPES, 10 Glucose and 0.33 NaH2PO4 (pH adjusted to 7.4 with concentrated NaOH). The intracellular solution used for Kv4.3 (Ito current) recordings was based on that from previous study of Kv4.3 2, 5 EGTA, 5 MgATP, 10 HEPES, 5.5 Glucose (pH adjusted to 7.3 with KOH). The intracellular solution for hERG (IKr current) recordings was similar to that used in prior studies from our laboratory 2, 5 EGTA, 5 MgATP, 10 HEPES (pH adjusted to 7.3 with KOH).The compound NS3623 was purchased from Santa Cruz Biotechnology Inc.  and dissolved in DMSO to a final concentration of 10 mmol/L. Individual aliquots were frozen at −20 °C and thawed for use on the day of the experiment. The DMSO stock  was diluted to a final concentration of 0.1% in extracellular solution to give an 10 μmol/L NS3623 solution, as used previously 2.4a) was always below 5 MΩ and series resistance was typically compensated by ∼40%. Voltage protocols are described in the “Results” section. Patch-Clamp recordings were analysed using Clampfit 10.3 . Statistics and graphs were prepared using Excel Professional Plus 2013  and Prism 7 for Windows .Patch pipettes (2.5–3 MΩ resistance) were fabricated from borosilicate glass capillaries  using a P-87 puller . Data were acquired and recorded with Clampex 10.3 software using an Axopatch 200 amplifier and Axon Digidata® 1322A . Data were digitized at 20 kHz during all voltage protocols and a bandwidth of 2 kHz was set on the amplifier. For whole-cell recordings, cells were continuously perfused with extracellular solution at 33 ± 1 °C. Access resistance  and analysis of variance or one-way ANOVA  when comparing three or more groups. Two-way ANOVA test (Sidak post-hoc test) was used when multiple comparison between different groups was necessary. In all cases, a p value of less than .05 was required for statistical confidence. Values are expressed as mean ± standard error of the mean (SEM).33.1m) in 10 mV increments from a holding voltage (Vh) of −80 mV, as illustrated in the inset to the top panel of toInitial experiments examined the effect of both KChIP2.2 and KChIP2.1 on Kv4.3. The voltage protocol involved square voltage steps from −60 to +40 mV  to +40 mV with varying interpulse intervals, Δt (10–2000 ms) as illustrated in the inset to rec) characterized the rate of recovery. The expression of KChIP2 isoforms decreased Kv4.3 τrec from 46.10 ± 6.19 ms (n = 18) to 5.88 ± 0.55 ms  and 11.99 ± 1.87 ms  in the presence of KChIP2.2 and KChIP2.1, respectively  by a factor of 3–4 (see Recovery of I 3–4 see . However 3–4 see .3.3hERG)) were evaluated with a standard hERG voltage clamp protocol V and slope parameters showed essentially no change with KChIP2.1/2.2 and DPP6  at each repolarization potential for each cell by fitting a bi-exponential function, giving fast (τfast) and slow (τslow) components . Itail amplitudes were then fitted by a monoexponential function to give time constants for activation which were compared across different expression conditions  (Activation time course of Inditions . Neithery ANOVA) .Fig. 5hEhERG current (during which a proportion of hERG channels inactivate), followed by a variable length repolarization step to −40 mV (between 2 and 20 ms long) to allow the channels to recover from inactivation. This was followed by a 20 ms test step to +40 mV to probe the extent of recovery from inactivation. Currents were normalized to the maximal current seen during the second +40 mV steps  KChIP2.2 and other kinetic interactions during the dynamic AP might produce some summative effect. We therefore examined potential modulation of IhERG by KChIP2.1/2.2/DPP6 accessory subunits under AP clamp as the most physiological stimulus, using a digitised human ventricular AP waveform as the voltage command hERG record from a cell expressing only hERG are shown in hERG current slowly increased during the plateau phase before quickly increasing in amplitude once the repolarization phase of the action potential started, with a maximal peak at ∼−40 mV Although KChIP2.1/2.2/DPP6 co-expression had only small effects on the individual kinetic parameters of I3.7to in ventricular cardiomyocytes, this compound also activates hERG ion channels in ex vivo preparations hERG and to explore the involvement of either KChIP2.1/2.2 isoforms and DPP6 in the compound-mediated response, we used our “standard” IhERG voltage step protocol (hERG traces in control (black line) and with 10 μmol/L NS3623 (dotted line) are shown in hERG tail amplitudes (at −40 mV) over time is shown in hERG is quickly, and reversibly, activated by NS3623. An increase in IhERG in response to NS3623 was always observed when KChIP2.1/2.2 and DPP6 were co-expressed and the degree enhancement of IhERG was independent of KChIP2.x and DPP6 co-expression  than Kv4.3 alone. Qualitatively, our results are in reasonable accord with previous studies assessing KChIP2.1 and Kv4.3 expressed in HEK 293 cells to inactivation follows a mono- or a bi-exponential time course Xenopus oocytes It is unclear whether I10 is ∼2 Co-expression of either KChIP2 isoform with DPP6 did not significantly change inactivation time constants compared to Kv4.3 alone, suggesting DPP6 opposes the kinetic increase mediated by KChIP2.x. A similar result has been previously reported for the KChIP2.1 isoform in oocytes 4.2hERG density hERG. The voltage and time-dependent properties of IhERG were generally unaltered although KChIP2.2 co-expression appeared to accelerate hERG activation  an increase in IhERG amplitude; (2) a rightward shift in the voltage-dependence of inactivation and (3) a slower onset of inactivation hERG. Since no accessory subunits were co-expressed with hERG in that study, their results support the idea of a direct interaction between NS3623 and hERG protein. Further studies in guinea pig (lacking Ito) showed NS3623 shortens the AP and decreases the appearance of extrasystoles in perfused hearts as well as reversing drug-induced QT prolongation, supporting the idea of some therapeutic potential for this compound to-modulating β-subunits do not influence the agonism of IhERG by NS3623, consistent with both the lack of modulatory effects of KChIP2/DPP6 on the channel and a direct α-subunit action.Hansen and colleagues characterized the effect of NS3623 on IXenopus oocytes, who failed to detect Kv4.3 activation Xenopus oocytes at room temperature). The concordance of our findings with the recent report by Calloe et al. to) highlights the importance of using mammalian cell expression systems for Kv4.3 studies. The rapid response to NS3623 which augments Kv4.3 current is consistent with acute effects of NS3623 on Ito leading to an increased epicardial action potential notch seen in canine left ventricular wedge preparations toIn relation to the effect of NS3623 on Kv4.3, our results show that the compound augments current amplitude without accessory subunit co-expression in contrast to findings by Hansen and colleagues using 4.4, modulating the interaction between KChIP3 and Kv4.3 Despite structural similarities, our data show that NS3623 binding sites must, in some ways, be different from the binding site(s) for NS5806 since stimulation of Kv4.3 current by NS5806 requires the presence of KChIP2 Medical Research Council U.K. (MR/N002903/1). JCH acknowledges a University of Bristol research fellowship.The study was funded by a programme grant from None.


More than 95% of the surviving single differentiated CD133–CD15–NESTIN– cell dedifferentiated into tumorigenic CD133+CD15+NESTIN+ GSCs, and this process was regulated by hypoxia inducible factor-1α. Moreover, the serum also played an important role in this dedifferentiation. These findings challenge the traditional glioma cell heterogeneity model, cell division model and glioma malignancy development model. Our study also highlights the mechanism of GBM recurrence and the importance of anti-hypoxia therapy. In addition to GSCs, residual differentiated tumor cells also substantially contribute to treatment resistance and the rapid, high recurrence of GBM.The standard treatment for Glioblastoma multiforme (GBM) is surgical resection and subsequent radiotherapy and chemotherapy. Surgical resection of GBM is typically restricted because of its invasive growth, which results in residual tumor cells including glioma stem cells (GSCs) and differentiated cells. Recurrence has been previously thought to occur as a result of these GSCs, and hypoxic microenvironment maintains the GSCs stemness also plays an important role. Summarizing traditional studies and we find many researchers ignored the influence of hypoxia on differentiated cells. We hypothesized that the residual differentiated cells may be dedifferentiated to GSC-like cells under hypoxia and play a crucial role in the rapid, high-frequency recurrence of GBM. Therefore, isolated CD133 Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults and Standard treatment comprises surgical resection followed by radiotherapy and chemotherapy. However, these strategies have minimal impact on extending the life expectancies of GBM patients , 2. To pet al [in vitro and in vivo. Using an in vitro approach, Li [Both GSCs and differentiated glioma cells are located in a hypoxic microenvironment , 7. For et al  demonstroach, Li  demonstrin vitro and tumorigenesis in vivo [2 [in vitro, as well as their tumorigenicity in vivo. Because hypoxia inducible factor-1a (HIF1α) is steadily expressed under hypoxic conditions and plays a significant role in angiopoiesis and stemness maintenance [Three basic features of GSCs are neurosphere formation, stemness marker expression  in vivo . We perf vivo [2 , and oxy vivo [2 ; thus, intenance , 13, we −CD15−NESTIN− glioma cell into neurosphere. Under hypoxia more than 60% cells survived at 3 days (d). Most surviving cells (95.38% ± 5.83 from GL261 and 99.10% ± 0.78 from U87) formed neurospheres at 21 d. However, under normoxia only 5.05% ± 1.12 from GL261 and 1.31% ± 1.32 from U87 cells formed sparse, irregular and non-adherent aggregates. Most surviving cells remained a single cell and died at 21 d . The protein levels of these markers sharply increased in hypoxia treatment cells and ranged from two- to six-fold higher compared with controls Figure . For the−CD15−NESTIN− glioma cells under hypoxia and detected the percentages of CD133, CD15 and NESTIN positive cells at days 0, 3, 6, 9, 12 and 15 with flow cytometry. The CD133 expression in primary glioma cells was 7.03% at day 0 (normoxia); however, its rate increased to 17.4% after hypoxia for 3 d and 62.9% after 12 d of hypoxia. The CD133 expression subsequently decreased but remained significantly increased compared with the control normoxia-treated cells. Similar results were identified in two other cell lines. The rate of CD15 positive U87 cells increased to 69.3% after 15 d of hypoxia and substantial changes were identified at hypoxia 12 d (59.055%). The CD15 expression was also significantly increased in hypoxia compared with the control in the GL261 and primary glioma cells, which increased from 1.152% to 44.226% and 8.77% to 39.9%, respectively. The NESTIN expression of U87 cells in control was only 2.252%. However, it was 35.15% at 3 d of hypoxia and then increased sharply to 93.1% at 15 d of hypoxia in a time-dependent manner. Similar results were identified in other two cell lines -induced neurosphere–derived cells  or normoxia (21%)-derived CD133−CD15−NESTIN− GL261-luc cells  were injected into the brains of adult female C57 mice. These mice were raised under normoxia (21% O2) as indicated in Figure 6. There was fewer tumor formation in the second group and the order of magnitude was even less than 104  underwent similar changes as the normoxia group in strategy 1, and group 4 was injected with differentiated CD133−CD15−NESTIN− Gl261-luc cells (3 × 104) but raised under 10% O2. At 25 d, tumors were detected in group 4 with magnitude orders greater than 106. However, the order of magnitude decreased to 4,161 in group 3  raised mice .Hypoxic microenvironment exists in glioma and this becomes more serious for residual glioma cells after surgery because of vascular damage , 25. It h Blazek  and Mathh Blazek  demonstr−CD15−NESTIN− cell was cultured under hypoxia for 21 days, more than 95% of surviving cells were induced to form neurospheres. Even if 20% of the single glioma cells were GSCs as reported [−CD15−NESTIN− differentiated cells were sorted through MACS three times. Therefore, the GSCs rate should be less than 20%. Another basic feature of GSCs is biomarker expression. CD133-positive cells as GSCs have been studied in many traditional studies [− cells may be tumorigenic in some cases [+ cell contamination as a result of missorting [in vivo [et al [in vivo. We used two different strategies to identify the tumorigenic ability of glioma cells under hypoxia exposure and found glioma cells derived from hypoxia exposure or mice injected with CD133−CD15−NESTIN− cells raised under hypoxia induced significant tumor formation with increased lethality and GSC markers expression. This in vivo study further demonstrated hypoxia dedifferentiated tumor cells into functional GSCs.GSCs are capable of neurosphere formation and clone proliferation , which ireported , 5, the  studies , 5, 28. me cases , 30. Altssorting , we fullssorting , NESTIN ssorting  and ABCGssorting , as wellssorting , 21. Botet al [0/G1 but a decrease in G2/M+S. Finally, Annexin-V assay revealed lower apoptosis tendency under hypoxia exposure. These results further demonstrated the induced neurospheres were stem cells, indicating single CD133−CD15−NESTIN− cell was successfully dedifferentiated into CD133+CD15+NESTIN+ GSCs by hypoxia and these neurospheres possessed the characteristics of stem cells.To further investigate the stem cell biology of these hypoxia induced cells, we employed asymmetric division , cell pret al  reportedIn CSC theory, researchers hypothesize CSCs divide asymmetrically, whereas differentiated cells grow symmetrically . HoweverRecent studies demonstrated there were many GSCs in the vascular niche, which are important to maintain the GSC phenotype and promote glioma malignant progression , 37, 38.Recently, additional studies have demonstrated stemness increased in different microenvironments, and transcription factors such as Sox-2, Oct-4, Klf-4 and Nanog were reprogrammed into both normal tissues including human dermal fibroblasts  and Müll−CD15−NESTIN− cells [8 cells/500 μl), followed by incubation for 15 min at 4°C with polyclonal rabbit anti-human CD133+ IgGs  or polyclonal rabbit anti-mouse CD133+ IgGs . After washing with PBS that contained 1% BSA, the cells were re-suspended in PBSE (108 cells/300 μl), labeled with goat anti-rabbit IgG MicroBeads , incubated for 15 min at 10°C, washed twice with PBSE and re-suspended in 500 μl PBSE. A cell separation column with a flow resistor was subsequently placed in a miniMACS magnet fixed on the MACS multistand and flushed with 500 μl PBSE. The 500 μl cell suspension was poured into the column reservoir. The CD133+ cells were retained within the magnet, and unlabeled nonmagnetic CD133− cells passed through the column and were collected. To increase the purity of the CD133− cells, we repeated the previous steps three times. The same methods were used to sort CD15− cells from CD133− cells and NESTIN− cells from CD133−CD15− cells. The CD133−CD15−NESTIN− cells were subsequently cultured in DMEM/F12+10% FBS at 37°C to maintain their differentiated status.GL261 and U87 cells, as well as primary glioma cells isolated from surgical waste were used as glioma cell lines. Magnetic cell sorting  was used to isolate CD133N− cells . In brie−CD15−NESTIN− glioma cells were diluted to 1,500 cells/1 ml DMEM/F12+10% FBS. 1 μl of medium was subsequently transferred to one well of a 96-well plate that had previously been pre-coated with 200 μl serum-free DMEM/F12 culture medium . Then we digested neurospheres to single cell suspension with 0.25% trypsin and added 1ml Trypan blue into the cell suspension and observed whether the colour of cell cytoplasm became blue. For asymmetric division, the neurospheres were subsequently transferred to six 24-well plates and half of the samples were cultured with serum-free medium that contained EGF and FGF2, and the other samples were cultured with DMEM/F12+10% FBS. All 24-well plates were incubated at 37°C with 21% O2 and 5% CO2 and recorded neurospheres morphology at 1, 3 and 5 d.The CD133m Figure . Three 9Newly formed neurospheres and differentiated cells after 48 h of hypoxia were fixed with 4% paraformaldehyde for 10 min at 4°C, washed with PBS and blocked with 10% normal serum for 20 min in PBS that contained 0.5% Triton X-100. The cells were incubated for 24 h at 4°C with primary antibodies against SOX-2 , OCT-4 , Nanog , KLF-4 , CD133 , CD15 , NESTIN , ABCG2 , VEGF  and HIF1α . Neurospheres and cells were washed three times with PBS for 5 min and then incubated at 37°C for 1 h with appropriate fluorophore-labeled secondary antibodies. Images were acquired with a laser scanning confocal microscope .Cells cultured in hypoxia for 0, 12, 24, 48 and 72 h were collected, subjected to SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk and incubated with antibodies against SOX-2 , OCT-4 , Nanog , KLF-4 , CD133 , CD15 , NESTIN , ABCG2 , VEGF  and HIF1α . Enhanced chemiluminescence was conducted for visualization.Cells exposed to hypoxia for 0, 3, 6, 9, 12 and 24 h were collected and used to examine the mRNA expression of stem cell markers with RT-PCR. The melting temperature was 94°C, 5 min; the denaturing temperature was 94°C, 30 s; the annealing temperature was 57°C, 30 s; the extending temperature was 72°C, 30 s; in total, 40 cycles were completed. The primer sequences were in −CD15−NESTIN− glioma cells exposed to hypoxia for 0, 3, 6, 9, 12 and 15d, HIF1α-ShRNA and HIF1α-vector glioma cells cultured under hypoxia 12d. Cells were collected and incubated with anti-CD133 antibody , anti-CD15 antibody  and anti-NESTIN antibody  and analyzed with FCM. For cell cycle and apoptosis analysis, cells cultured in DMEM/F12 with 1%FBS were exposed to normoxia and hypoxia for 0, 1, 3, 5 and 7d; and then collected and detected for FCM analysis.Flow cytometry (FCM) was used to determine the expression of GSC markers CD133, CD15 and NESTIN in CD1332) and others exposed to hypoxia (1% O2). Cell growth was examined on day 1, 3, 5, 7, 9 and 11, respectively. 10μl of CCK-8 solution and 90μl culture medium were mixed and added into each well. The resulting samples were cultured for 2 h respectively under hypoxia or normoxia condition. The absorbance at 450nm was measured with ELISA reader .CCK-8 was used to examine cell growth. Logarithmic phase glioma cells were collected and digested to prepare single cell suspension. The cells were counted and seeded into four 96-well plates (2000 cells/well). Two plates were cultured with normoxia . The primers were used to amplify the luciferase gene and for sequencing.upstream 5′-GAAGATCTCACCATGGAAGACG CCAA-3′downstream 5′-GCGTCGACTTACACGGCGATC TTTCCGC-3′The sequenced plasmid was cut by the restriction enzymes. The fragment was sub-cloned into the plenti6-V5-D-TOPO plasmid and packaged into virus. The target Gl261 cells were subsequently infected with virus for 2 days and screened using blasticidin; stably infected Gl261-Luc cells were obtained at approximately 2 weeks.in vivo.We used two different strategies Figure  to inves3 differentiated CD133−CD15−NESTIN− GL261-Luc cells were seeded into each well of 6-well plates that had previously been pre-coated with 2 ml serum-free DMEM/F12 culture medium and cultured under hypoxia conditions 14d; then collected and digested neurospheres with 0.25% trypsin to single cell suspension. Counted and injected 104 cells into the right frontal lobe of 6-week-old C57 female mice (n = 25) that were raised under normoxia. Fifteen mice were used to examine the survival rate, and the remaining mice were used to investigate tumor growth by the bioluminescence of cancer cells at 5, 10, 15, 20 and 25 d. To determine the amount of bioluminescence, 200 μl (15 mg/1 ml) of luciferins were administered via peritoneal injection to mice under anesthesia, and the bioluminescence was detected using the NightOWL Macro Imaging system . For control group, cells cultured under normoxia (21% O2) were injected into the same brain region of the mice (n = 21) and examined using identical procedures.Strategy 1: 5 × 104 CD133−CD15−NESTIN− GL261-Luc cells exposed to normoxia into age-matched C57 mouse brains. The mice were raised under normoxia  or hypoxia . On 5, 10, 15, 20 and 25 d, bioluminescence was measured as previously described.Strategy 2: We injected 3 × 102 or 10% O2 without tumor cells. Immunohistochemistry, RT-PCR and Western blot were used to examine the stem cell markers and HIF1α expression.All animals were raised for 30 days, and the 30 day survival rate was assessed. To rule out the influence of other factors on mice, 30 additional mice were raised in 21% O2; the numbers of surviving cells and newly formed neurospheres were recorded at 0, 3, 7, 14 and 21 d. Cell death rate was examined at 1, 3, 5 and 7 d.Cell suspensions were diluted to 1,500 cells/1 ml DMEM/F12+10% FBS or 1,500 cells/1 ml DMEM/F12 Figure . Single −CD15−NESTIN− glioma cells were counted, and 105 CD133−CD15−NESTIN− cells were seeded into 6-well plates. HIF1α-ShRNA-lentivirus (MOI=1) was added to 6-well plates following cell attachment for 24 h. 1 μg/ml of puromycin was subsequently added to the 6-well plates following lentiviral infection for 48 h. This process lasted one week. The cells were then collected, and HIF1α expression was detected via Western blot. Successful HIF1α-interference cells were cultured in DMEM/F12+10% FBS to maintain growth. Digoxin  was dissolved at 100 mM in DMSO, and the effective concentration was 100 nm/L [in vivo study, we implanted 3 × 104 HIF1α-ShRNA CD133−CD15−NESTIN− GL261 cells , HIF1α-Vector CD133−CD15−NESTIN− GL261 cells  and CD133−CD15−NESTIN− GL261 cells  respectively into C57 mice brain. Fifteen mice of each group were used to examine survival rate, and the remaining mice were used to investigate tumor growth by the bioluminescence of cancer cells at 20 d as identical procedures above.The HIF1α-ShRNA lentivirus was purchased from Cyagen, Guangzhou, China. CD133T test and one-way analysis of variance (ANOVA) when necessary. A log-rank test was used to assess the survival rate and P < 0.05 was considered statistically significant.SPSS 19.0 was used for statistical analysis. Data were expressed as the means ± standard deviations (SDs). Differences were evaluated with


We present an in-depth experimental study of a new class of heterometallic, redox-switchable single-molecule magnets (SMMs). 3+ (oblate) and Er3+ (prolate) which can be reversibly oxidized by one electron to yield their respective charge neutral redox partners . Importantly, compounds 1 and 2 are thermally stable which allowed for detailed studies of their magnetization dynamics. We show that the Dy3+–[1]/1 system can function as an “on”/“off” or a “slow”/“fast” redox switchable SMM system in the absence or presence of applied dc fields, respectively. The Er3+ based –[2]/2 system features “on”/“off” switchability of SMM properties in the presence of applied fields. Results from electrochemical investigations, UV-vis-NIR spectroscopy, and 57Fe Mössbauer spectroscopy indicate the presence of significant electronic communication between the mixed-valent Fe ions in 1 and 2 in both solution and solid state. This comparative evaluation of redox-switchable magnetization dynamics in low coordinate lanthanide complexes may be used as a potential blueprint toward the development of future switchable magnetic materials.Single-molecule magnets (SMMs) are considered viable candidates for next-generation data storage and quantum computing. Systems featuring switchability of their magnetization dynamics are particularly interesting with respect to accessing more complex logic gates and device architectures. Here we show that transition metal based redox events can be exploited to enable reversible switchability of slow magnetic relaxation of magnetically anisotropic lanthanide ions. Specifically, we report anionic homoleptic bis-diamidoferrocene complexes of Dy Ueff) to reorientation of their molecular spin.via ligand field considerations. While initial efforts in the field focused on slow magnetic relaxation in multi-nuclear transition metal complexes,Ueff.Single-molecule magnets (SMMs) have been attracting considerable attention for potential applications in quantum computing, high-density data storage and molecular spintronics.via external stimuli is an essential aspect. Possible stimuli include light, temperature, pressure, chemical, dc field, and electric potential.3+ phthalocyanine double-decker complexes, are redox-switchable SIMs, undergoing changes in magnetization dynamics and hysteretic behaviour upon electrochemical generation of an open shell π-system.With the ultimate-goal of utilizing SMMs in devices, the development of methodologies that will allow for the control of dynamic magnetic properties reversibly There have been several reported examples of redox-switchability in multinuclear transition metal and lanthanide based SMMs. In these systems, the oxidation and/or reduction of either a ligand3+ and Dy3+ double-decker lanthanide(iii) phthalocyanine complexes,iii)-based SIM. In that example an intramolecularly attached Ru2/3+ redox switch was utilized to modify the magnetic relaxation dynamics of a Dy3+-based SIM.2+ to Ru3+ was found to enhance slow magnetic relaxation, either through perturbations of the ligand field or the addition of another spin-carrier to the system. However, this interesting system suffered from limited thermal stability at room temperature.Aside from the Tb2, make it an attractive moiety to include in complexes for redox switchability applications.iii) ion. Previously, Diaconescu et al. reported a homoleptic uranium(iv) compound stabilized by two bidentate diamidoferrocene ligands.iii) bis-diamidoferrocene compounds.The reversible redox properties of ferrocene, FeCp3+ ion-based redox switchable SMMs using the redox chemistry of ferrocene/ferrocenium in the ligand scaffold.2+ to Fe3+ in Dy3+ (oblate electron density) and Er3+ (prolate electron density) bis-diaminoferrocene compounds modulates dynamic magnetic properties. Depending on the experimental conditions, these materials can exhibit switchability of their slow magnetic relaxation either between “on” and “off” or between “slow” and “fast”. Remarkably, this is the first example of redox switchable SMM properties observed in an Er3+ compound. Additionally, this is the first magnetic investigation of homoleptic, four-coordinate Dy3+ and Er3+ complexes. This set of compounds has been characterized using X-ray crystallography, dc/ac magnetometry, 57Fe Mössbauer spectroscopy, UV-vis-NIR spectroscopy and cyclic voltammetry.Herein, we present the first class of LnNotably, this is the first example of using the redox chemistry of a transition metal to alter the magnetization dynamics of a lanthanide ion, while maintaining thermal stability of all redox partners. This molecular level study is intended to provide design guidelines for future switchable solid materials.3 (Ln = Dy or Er) and two equivalents of [K2(OEt2)]fc[NSi(t-Bu)Me2]2 in thf yielded the anionic lanthanide(iii) species K(thf)5[Ln(fc[NSi(t-Bu)Me2]2)2]  (5[Ln(fc[NSi(t-Bu)Me2]2)2] with half an equivalent of iodine afforded the neutral mixed valent Fe3+/Fe2+ compounds Dy(fc[NSi(t-Bu)Me2]2)21 and Er(fc[NSi(t-Bu)Me2]2)22 (1) and dark red-brown (2) block crystals were grown from concentrated hexanes solutions at –30 °C over 24 h.The reaction between LnI = [2]–) . Yellow Me2]2)22 . Dark pu–[1] and –[2] readily crystallize under the experimental conditions, single crystal X-ray diffraction data indicated severe disorder. However, the connectivity of –[1] and –[2] could be established and equivalent distances between the Ln3+ ion and the two Fe2+ ions were found. The neutral, mixed-valent compounds 1 and 2 are isostructural to each other and crystallize in the orthorhombic space group Pbca ° to 131.9(2)° and 102.2(6)° to 126.7(6)° for 1 and 2, respectively. The differences in Fe–C bond lengths of the two crystallographically independent Fe sites indicate valence localization in the solid state at this temperature (110 K). The two Ln···Fe distances in 1 and 2 are inequivalent, with long Ln···Fe distances of 3.792(2) Å and 3.819(5) Å and short Ln···Fe distances of 3.368(2) Å and 3.498(4) Å , 2.338(4) Å and 2.29(1), 2.27(2) Å) and shorter distances (2.262(4), 2.252(4) Å and 2.22(2), 2.21(2) Å). In the solid state, the closest Fe···Fe distances are intermolecular with separations of 6.464(2) Å and 6.478(5) Å, while the intramolecular Fe···Fe distances are 7.098(2) Å and 7.267(5) Å for 1 and 2, respectively . Upon cooling, the χMT decreases gradually, then more rapidly below 130 K to reach a minimum of 12.55 cm3 K mol–1 at 2 K. This decrease is mainly attributed to the thermal depopulation of excited states. After one-electron oxidation of –[1] to form 1, an increase in the room temperature χMT value to 14.36 cm3 K mol–1 is observed. This increase is in accordance with the presence of a single Dy3+ ion and an additional non-interacting S = 1/2 site . Upon decreasing the temperature, the χMT value of 1 decreases more rapidly than –[1] below 60 K, reaching a minimum of 10.13 cm3 K mol–1 at 2 K. The more rapid decrease could be indicative of thermal depopulation of Stark sublevels or the presence of intra- and/or inter-molecular antiferromagnetic interactions.47To investigate static magnetic properties, direct current (dc) magnetic measurements were carried out using crushed polycrystalline samples under an applied field of 1000 Oe from 300 K to 2 K . The roo3+ analogues, the room temperature χMT value for –[2] is 10.82 cm3 K mol–1. This value is only slightly lower than the expected value of 11.28 cm3 K mol–1 for a single isolated Er3+ ion . After one-electron oxidation to 2, the room temperature χMT value increases to 11.43 cm3 K mol–1, corresponding to one Er3+ ion and one non-interacting Fe3+ ion. As observed in the analogous Dy3+ compounds, there is a more rapid decrease in the χMT of the oxidized mixed-valent compound 2 at low temperatures.For the ErM) was measured for each compound with fields up to 70 kOe (7 T) over a temperature range of 2–8 K  suggests the presence of significant magnetic anisotropy in –[1]  of the ac susceptibility was observed under zero applied dc field, indicative of slow magnetic relaxation (χ′) vs. out-of-phase (χ′′) components of the ac susceptibility were fit to a generalized Debye modelτ , a relaxation process that proceeds without the input of thermal energy.Dynamic magnetic properties were investigated using alternating-current (ac) measurements. In laxation . Cole–Coe modelτ . The natius plot . The lar–[1] , τ0 , and Ueff  were treated as free-fit parameters. For Kramers ions, parameter n2 in the Raman pathway is expected to be 9; although allowing n2 to be a free-fit parameter resulted in a value of n = 5. A value lower than 9 may be expected in systems with low-lying excited states if optical phonons are taken into consideration.τ vs. T–1.The parameters Ueff = 27.3(8) cm–1 (τ0 = 1.63(2) × 10–6 s), for the thermally activated Orbach process was calculated for –[1] under Hdc = 0 Oe using eqn (1). All parameters of the fit are listed in Table S5.Due to the predominance of quantum tunnelling of magnetization (QTM) at low temperatures, few temperature dependent points were obtained. However, a barrier height, 1 shows no evidence of slow relaxation under zero applied dc field at ac frequencies up to 1000 Hz  likely make the largest contribution to the change in magnetization dynamics. The appreciable loss of symmetry upon oxidation and change in ligand field would undoubtedly influence the orientation of the magnetic anisotropy axis and therefore the magnetization dynamics. In the absence of applied dc fields, the changes in magnetization dynamics upon reversible one-electron oxidation of –[1] to 1 can be thought of as “on”/“off” switching of the slow magnetic relaxation (frequencies up to 1000 Hz)  .3+ compounds, –[2] and 2, no signal was observed in the out-of-phase component (χ′′) of the ac susceptibility under zero dc field at ac frequencies up to 1000 Hz  observed within the ac frequency range of 1 to 1000 Hz  are shifted to lower frequencies relative to the zero field measurements, as the reduction of QTM results in slower magnetic relaxation. Furthermore, with the application of the 1000 Oe dc field, the Arrhenius plot displays temperature dependence over the entire temperature regime  for the applied field measurements were fit using least squares regression to a model that accounted for multiple relaxation pathways, including direct, QTM, Raman, and Orbach (eqn (2)).Arrhenius plots .n1 = 4 for Kramers ions in the absence of hyperfine interactions.D was added to account for the field independent contributions from Raman and Orbach relaxation processes.A, B1, B2, and D were treated as free fit parameters. All fitting parameters are listed in Tables S5 and S6.–[1] at 5 K, the very gradual decrease in τ values at fields above 1000 Oe suggests minimal contributions of single phonon direction relaxation mechanisms , according to eqn (2). For –[1], the temperature dependence of τ was fit to direct, Raman, and Orbach relaxation processes  cm–1 (τ0 = 7.3(7) × 10–7 s) was obtained; 18.7 cm–1 larger than the barrier calculated under zero dc field.The parameters obtained from eqn (3) were held fixed while fitting the temperature dependent Arrhenius plots   occurs at a field of 1750 Oe at 2 K. However, to maintain consistency with the ac measurements for –[1], variable temperature ac measurements for 1 were carried out using a 1000 Oe dc field  cm–1 (τ0 = 5.0(4) × 10–7 s)  . A valuedc field . The maxmpound 1 . The Arr 10–7 s) . Notably–[1] and 1  (Ueff = 29(2) cm–1 (τ0 = 4(1) × 10–7 s). The one-electron oxidized species, 2, did not display any signs of slow magnetic relaxation under dc fields as high as 1500 Oe in ac experiments  . Variabl eqn (3) . The cor eqn (3) , accordi Fig. S30.3+ ion in the –[2]/2 system allows solely for reversible “on”/“off” switching of the slow magnetic relaxation    , while tc field)  and “sloc field)  and 7.3+ (oblate) and Er3+ (prolate) result in very different magnetic anisotropy axes and therefore different magnetization dynamics under the same ligand field and molecular symmetry conditions. Previously, isostructural Dy3+ and Er3+ complexes have been shown to exhibit different magnetization dynamics. In the four-coordinate, trigonal pyramidal series of compounds [Li(thf)4][Ln{N(SiMe3)2}3Cl]·2 thf, the Er3+ analogue displayed SMM behaviour under zero dc field, whereas the Dy3+ analogue exhibited only field induced SMM behaviour.3+.59The distinct shapes of f-electron density of Dy3+ ion than for the prolate Er3+ ion. The differences in behaviour between the Dy3+ and Er3+ complexes are attributed to the orientation of the anisotropy axis under the same ligand field conditions due to the difference in f-electron density. We believe the largest contributor to the change in magnetization dynamics upon oxidation to be the change in ligand field and lowering of local symmetry. In detail, the oxidation of one of the diamidoferrocene ligands (in going from –[1] and –[2] to 1 and 2) results in inequivalent binding of the two ligands to the central lanthanide ion. Additionally, packing effects and the presence or absence of counter ions may contribute to the structural changes.The coordination geometry of the bis-diamidoferrocene complexes presented here was found to be relatively more favourable for the oblate Dy1 was determined utilizing a quantitative electrostatic model for the prediction of the orientation of the ground state anisotropy axis in Dy3+ compounds described by Chilton et al. (MAGELLAN).1, the anisotropy axis was determined under three different scenarios: (1) assigning both Fe centres as neutral, (2) assigning a +1 charge to the Fe centre closer to the Dy3+ ion, and (3) assigning a +1 charge to the Fe centre further from the Dy3+ ion .f-block element is indeed critically involved in the electronic communication between the mixed-valent iron ions, resulting in larger electronic coupling for the actinide ion (U4+) than for the trivalent lanthanide ions , featuring the more contracted frontier orbitals.The separation in electrochemical potentials between individual redox couples of multi-redox systems can provide a useful initial estimate for the presence of electronic communication in mixed-valent species. The cyclic voltammogram of a solution of 57Fe Mössbauer spectroscopy to establish the presence of electronic communication of the mixed-valent iron ions in 1 in the solid state. In the 57Fe Mössbauer spectrum of the all ferrous species –[1] in zero field, a single characteristic doublet was observed, corresponding to the spectroscopically identical low-spin Fe2+ ions in –[1], with an isomer shift (δ) of 0.54 mm s–1 and quadrupole splitting (ΔEQ) of 2.34 mm s–1  and an equally contributing second doublet corresponding to the Fe3+ ion . The Mössbauer spectrum is consistent with a trapped valent system over the temperature range studied . However, electronic communication between Fe2+ and Fe3+ is clearly present, as the signal corresponding to the ferric site is quadrupole split. In the absence of electronic communication this signal would be expected to occur as a singlet.EQ observed for the ferrous site in 1 as compared to –[1] is also consistent with the formulation of Fe2+···Fe3+ electronic communication. Similar spectroscopic signatures in mixed-valent dinuclear iron complexes have previously been reported for [Cp*2Fe2(as-indacene)]˙+.62We utilized  Fig. S36. The 57Fdoublets . The spedoublets  model  transition was observed in the near-IR region of dilute thf solutions of the mixed-valent compounds 2+/3+ redox couples can be exploited to alter magnetization dynamics of Dy3+ and Er3+ based SMMs. The mixed valent Fe ions in complexes 1 and 2 feature electronic communication with each other. Importantly, redox-switchable “on”/“off” and “slow”/“fast” SMM behaviour can be obtained depending on the experimental conditions and the nature of the four-coordinate lanthanide ion. The combined results of this comprehensive molecular level study are important contributions towards the development of rational molecular design guidelines for future switchable magnetic molecules and materials.Our studies highlight the utility of diamidoferrocene ligands in the construction of redox switchable SMMs. The high chemical reversibility of the Feiii) iodide and iodine were purchased from Alfa Aesar. Eicosane was purchased from Acros Organics. Sublimed anhydrous erbium(iii) iodide was generously donated by Prof. Tim Hughbanks' group (Texas A&M). fc[HNSi(t-Bu)Me2]2 was prepared as previously described.via the deprotonation of toluene by nBuLi/KOtBu as described previously.All reactions and manipulations were carried out under anaerobic and anhydrous conditions in an argon filled glovebox (Vigor). All syntheses and manipulations were carried out using disposable plastic spatulas. Tetrahydrofuran (thf), hexanes, toluene and diethyl ether were dried and deoxygenated using a solvent purification system (JC Meyer Solvent Systems) and were stored over molecular sieves in an argon-filled glovebox. Anhydrous dysprosium(via a modification of a previous procedure.t-Bu)Me2]2  was dissolved in diethyl ether (8 ml) and cooled in a freezer (–30 °C) for 30 min. Solid benzyl potassium  was added to the cold stirring solution of fc[HNSi(t-Bu)Me2]2 in diethyl ether. The reaction mixture was allowed to warm up to rt and stirred for 2 h. All solvent was removed in vacuo. The remaining orange solid was washed with hexanes and collected on a medium frit. Solid was dried in vacuo and used without further purification .Performed 2(OEt2)]fc[NSi(t-Bu)Me2]2  was dissolved in 3 ml of tetrahydrofuran and cooled in the freezer (–30 °C) for 30 min. To a separate vial, dysprosium(iii) iodide  and thf (3 ml) were added and the solution was cooled in the freezer (–30 °C) for 30 min. The [K2(OEt2)]fc[NSi(t-Bu)Me2]2 solution was added dropwise to the stirring DyI3 suspension while cold. The reaction mixture was stirred for 1.5 h at rt. All volatiles were removed in vacuo and the solid was washed with hexanes and dried. A minimal amount of thf was added and the solution was filtered through Celite. Removal of the thf in vacuo yielded a yellow-orange solid. Air sensitive yellow-orange plate crystals were obtained from a concentrated thf solution layered with hexanes at –30 °C for 24 h . Unit cell parameters (110 K): a = 16.500(2) Å, b = 19.510(3) Å, c = 12.286(2) Å, α = 90°, β = 90°, γ = 90°, V = 3955 Å3. Anal. calcd for C64H116N4DyFe2KSi4O5 (–[1]): C, 53.11; H, 8.08; N, 3.87. Found: C, 52.62; H, 7.77; N, 3.59.[K–[1], with [K2(OEt2)]fc[NSi(t-Bu)Me2]2 , erbium(iii) iodide  and 8 ml of tetrahydrofuran. Single crystals were obtained from a concentrated thf solution layered with hexanes in the freezer (–30 °C) for 24 h  of very air-sensitive yellow-orange plate crystals. Unit cell parameters (110 K): a = 16.443(3) Å, b = 18.088(4) Å, c = 12.828(3) Å, α = 90°, β = 90°, γ = 90°, V = 3815 Å3. Anal. Calcd for C56H100N4ErFe2KSi4O3 (–[2] – 2 thf): C, 51.4; H, 7.71; N, 4.28. Found: C, 51.32; H, 7.50; N, 3.86.Same procedure as in the synthesis of 5[Dy(fc[NSi(t-Bu)Me2]2)2]  and diethyl ether (5 ml) were added to a vial and placed in the freezer (–30 °C) for 30 min. To a separate vial, iodine  and diethyl ether (5 ml) were added and placed in the freezer (–30 °C) for 30 min. The iodine solution was added to the stirring K(thf)5[Dy(fc[NSi(t-Bu)Me2]2)2] solution while cold, resulting in immediate colour change to dark red. The reaction mixture was stirred for 40 min at rt. All volatiles were removed in vacuo. The resulting dark solid was extracted into hexanes and filtered through Celite. The volatiles were removed in vacuo, yielding a dark purple solid. X-ray quality dark red-purple block crystals were grown from a concentrated hexanes solution after 24 h at –30 °C . Unit cell parameters (110 K): Pbca, a = 20.025(4) Å, b = 19.663(4) Å, c = 24.499(5) Å, α = 90°, β = 90°, γ = 90°, V = 9646 Å3. Anal. calcd for C44H76N4DyFe2Si4 (1): C, 50.44; H, 7.31; N, 5.35%. Found: C, 50.17; H, 6.88; N, 5.30%.K(thf)1 was followed using K(thf)5[Er(fc[NSi(t-Bu)Me2]2)2] , iodine  and diethyl ether (10 ml). X-ray quality air sensitive dark red-orange block crystals were grown from a concentrated hexanes solution after 24 h at –30 °C . Unit cell parameters (110 K): Pbca, a = 19.794(2) Å, b = 19.822(2) Å, c = 24.603(2) Å, α = 90°, β = 90°, γ = 90°, V = 9653 Å3: Anal. calcd. for C44H76N4ErFe2Si4 (2): C, 50.22; H, 7.28; N, 5.32%. Found: C, 49.99; H, 7.24; N, 5.20%.The same procedure as for 2 stream (Oxford) maintained at 110 K. A BRUKER APEX 2 Duo X-ray (three-circle) diffractometer was used for crystal screening, unit cell determination, and data collection. The X-ray radiation employed was generated from a Mo sealed X-ray tube . Bruker AXS APEX II software was used for data collection and reduction. Absorption corrections were applied using the program SADABS.F2) to convergence.1, the Si4(C39–C44) group was found disordered between two positions and was modeled successfully with an occupancy ratio of 0.55 : 0.45.Crystals suitable for X-ray diffraction were mounted on a nylon loop and placed in a cold Nvs. H plots were fit using least squares regression.A representative procedure for the preparation of the samples for magnetic characterization is as follows. Crystalline sample was crushed up into a fine powder before loading into a high purity 7 mm NMR tube (Norell). A layer of eicosane was added to the tube, covering the sample. The tube was then flame sealed under vacuum. To restrain the sample, the sealed tube was placed in a water bath (39 °C) until the eicosane melted and was evenly distributed throughout the sample. The sample was loaded into a straw affixed to the end of the sample rod. Magnetic measurements were carried out using a Quantum Design MPMS 3 SQUID magnetometer (TAMU Vice President of Research). Dc susceptibility measurements were carried out over a temperature range of 1.8 to 300 K. Ac measurements were carried out using a 2 Oe switching field. Data was corrected for diamagnetic contributions from the straw, sample tube, eicosane and core diamagnetism using Pascal's constants.Crystalline samples were loaded into a Teflon cup inside a glovebox and covered with a layer of paraffin oil. The cup was brought out of the glovebox and immediately stored frozen in liquid nitrogen until measured. Mössbauer spectra were collected on a model MS4 WRC low-field, variable temperature spectrometer . Zero magnetic field spectra were obtained by removing the 500 G magnets from the exterior of the instrument. Temperatures were varied using a temperature controller on the heating coil on the sample holder. The instrument was calibrated using an α-Fe foil at room temperature. Obtained spectra were fitted using WMOSS software (See Co.).–1 to 250 mV s–1 were used. Ferrocene was added at the end of each data collection and the ferrocene/ferrocenium couple was used as an internal standard.Cyclic voltammograms were measured in an argon filled glovebox (Vigor). Data were collected using a Gamry Instruments Reference 600 potentiostat with Gamry Framework software. Glassy carbon working electrode, 1 mm diameter Pt wire counter electrode, and silver-wire pseudo-reference electrode were used. Scan rates of 100 mV sThere are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.


Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension.Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with  The topological entrapment of chromosomes by cohesin  is central to genome integrity124578911121314Eco1 is an acetyltransferase that belongs to the Gcn5-related N-acetyltransferase (GNAT) family1618192223Cdc4-dependent ubiquitination and degradation after S phaseXenopus laevis, onto chromatin was dependent on the pre-RC assembly but independent of cohesin loading and DNA synthesisAs well as its function in establishing replication-coupled cohesion, Eco1 has also been proposed to act during double-strand break repair. Break-induced phosphorylation of Scc1 is thought to trigger its Eco1-mediated acetylation, which also antagonizes Wapl releasing activityLike cohesin and its regulatory subunits, Eco1 homologues also have important implications in human developmental disorders2831in-vitro acetylation assay and coupled it with mass spectrometry (MS) analyses to determine both the dynamics and substrate specificity of Eco1-mediated acetylation. In our assays, we observe a fast acetylation event of K112 occurring prior to the acetylation of K113. By aligning the sequences of different acetylated cohesin peptides, we show that the target lysine of acetylation is favoured by the flanking of an aliphatic residue and an acidic/polar residue, and that this motif can be recognized in a bidirectional fashion. Furthermore, we present the crystal structures of Xenopus laevis Eco2 (xEco2) ACT domain bound to two different substrate peptides, which reveal that Eco1 substrate specificity is determined by the concerted conformational changes of the conserved central β hairpin and the C-terminal extension (C extension).Despite the importance of acetylation in cohesion establishment and human diseases, little is known about how Eco1 targets its canonical substrate Smc3 and how this acetylation stabilizes cohesin on DNA. To provide further insights into substrate recognition by Eco1, we reconstitute an S. cerevisiae K112/K113; human K105/106) in Smc3 -lysine motifs are also substrates for deacetylation by the Sirtuins, Sirt1-3 where a similar position-dependent rate dependence is observed and the acetylation state of one lysine may affect the rate of the adjacent deacetylationin-vitro Eco1-mediated acetylation with recombinantly purified cohesin core complex and regulatory subunits  and subjected the reaction products to MS analysis . To gain insights into Smc3 recognition by Eco1, we performed crystallographic studies on the Xenopus laevis Eco2 (xEco2) ACT domain (residues 523–702) (2745The variation in acetylation rates seen in our MS data  is likel523–702) , with an702) 2745. Crystal702) 2745.S. cerevisiae W216G; human W539G)  (PDB: 4MXE)n W539G) . Mutatio, of the hydrophobic pocket in yeast had little impact of Smc3 acetylation, combining both mutations or deleting the entire β hairpin (residues 189–198) drastically reduced yeast survival and eliminated Smc3 tandem lysine acetylation, confirming the importance of the β hairpin in Eco1 substrate recognition en in MS  and the en in MS .in-vitro acetylation assays coupled with mass spectrometry. We discover Eco1 substrate motif consensuses and the acetylation of Smc3 K112 to be a faster event prior to the slower but biologically important K113 acetylation. Furthermore, our xEco2-substrate structures illustrate that substrate specificity is conferred by the orchestrated conformational changes of the conserved central β hairpin and the C extension , Scc2-Scc4 complex, Pds5, and Wapl were amplified by polymerase chain reaction (PCR) using genomic DNA as templates and cloned into a modified pFBDM vector with a double Strep-tag II (ds) and a tobacco etch virus (TEV) cleavage site at the N terminus of Rad21, the C terminus of Scc2, the C terminus of Pds5, and the C terminus of Wapl respectively. The resultant protein expression cassettes were recombined with the DH10MultiBac cells to create the corresponding bacmids. S. cerevisiae cohesin was expressed using the baculovirus/insect cell (Sf21) systems whereas the Scc2-Scc4, Pds5, and Wapl were expressed using Hi5 insect cells. All proteins were purified by Strep-Tactin (Qiagen), anion exchange chromatography Porous Q, and S200 size-exclusion chromatography .S. cerevisiae subunits: Scc2-Scc4, Pds5, and Wapl for 90 minutes at 37 °C. The reaction was carried out in 50 mM Tris pH 8, 50 mM NaCl, 0.5 mM DTT, 5 mM MgCl2, and 1.5 mM Acetyl-CoA (Buffer A). 70 μM Eco1 was also incubated with 0.7 μM of Smc1-Sm3-Scc1-Scc3 in Buffer A with an addition of 1 mM ATP. The reactions were stopped and samples were analyzed by SDS-PAGE. The resulting protein bands were visualized by Coomassie blue staining and cut for MS analysis. To obtain more information on the tandem acetylation of Smc3’s K112 and K113, a time course experiment was conducted with Smc1-Smc3-Scc1-Scc3 in Buffer A with an addition of 1 mM ATP. Samples were taken at 0, 10, 20, and 40 minutes.To obtain a consensus motif, Eco1 at a concentration of 70 μM was incubated with 0.7 μM of the following cohesin 3) and destained in 10 mM triethylammonium bicarbonate (TEAB), 40% acetonitrile at 37 °C. The solution was removed and the gel plugs were dehydrated with acetonitrile. 4 μl of 4% (v/v) formaldehyde (aq) and 4 μl 0.6 M sodium cyanoborohydride (aq) was added to 100 μl 10 mM TEAB, and transferred to the gel pieces for 1 h at 22 °C. The solution was removed, and the gel pieces were twice washed with 50 μl 1% ammonium hydroxide to quench any further reaction, after each wash the gel pieces were dried with acetonitrile. 50 μl 5% formic acid was then added to the gel pieces, removed and dried with acetonitrile. The gel pieces were washed four times in 10 mM TEAB and dried in acetonitrile.Gel pieces were cut into small gel plugs . The samples were analyzed using PRM , with one full MS scan  being completed followed by six separate MS/MS events  at m/z 574.31, 578.97, 583.63, 860.97, 867.95, and 874.94; this sequence was repeated throughout the total analysis. PRM chromatograms were then extracted using Skyline 3.5.0.9319 software.A Thermo Scientific Q Exactive mass spectrometer coupled to an UltiMate 3000 HPLC system for on-line liquid chromatographic separation was used for data acquisition. Each sample was analyzed in triplicate (10 μl per injection) and each LC-MS/MS run consisted of a 1 h gradient elution .txt output file was used for further analysis using Perseus software. Acetylated peptides of cohesin subunits were categorized manually as forward (ϕ-AcK-B/Z) (n = 36), reverse (B/Z-AcK-ϕ) (n = 42), and no direction (n = 27). Eco1 substrate motif consensuses were subsequently generated using the iceLogo server6 tag and a TEV protease site. This vector was transformed using standard procedures into BL21(DE3)-RIL competent cells (Agilent). 1L of LB was inoculated with an overnight culture of cells grown at 37 °C. The 1L culture was grown until an OD of 0.6 was reached at which point, the cells were cooled to 16 °C. The expression xEco2 was induced by the addition of 0.25 mM IPTG for 18 hours. The harvested cells were sonicated in 50 mM Tris 8.5, 300 mM NaCl, 0.5 mM TCEP, 10% Glycerol and 10 mM imidazole and then centrifuged at 35000 × g, 4 °C for 1 hour. The following supernatant was loaded onto a 5 mL His-Trap HP  and the protein was eluted in a gradient of imidazole from 10 mM to 0.5 M. The protein containing fractions were loaded onto a pre-equilibrated MonoQ 10/300 GL  column in 20 mM Tris 8.5, 50 mM NaCl and 0.5 mM TCEP. xEco2 was eluted from the ion exchange in a 50 mM to 1M NaCl gradient. The protein peak was loaded onto a HiLoad 16/60 Superdex 75 column  equilibrated in 10 mM Tris 7.5, 150 mM NaCl and 0.5 mM TCEP. SDS-PAGE analysis was used to determine protein purity.xEco2 ACT domain was amplified (residue 523–702) and cloned into a pET-28a vector, which contained the addition of an N-terminal His2O. After 4 h, the resin was removed by filtration and peptides were precipitated with diethyl ether on ice. Peptides were isolated by centrifugation, dissolved in H2O and freeze dried overnight. Peptides were purified on a C8 reverse phase HPLC column . Buffer A was 1% acetonitrile, 0.08% trifluoroacetic acid in H2O, buffer B was 90% acetonitrile, 0.08% trifluoroacetic acid in H2O. The elution gradient was from 10% to 50% buffer B, over 40 min at a flow rate of 8 ml/min. Conjugation of the purified bromopropionyl-peptide to Coenzyme A was achieved by adding 25 mg (5 eq) of Coenzyme A hydrate (Sigma) dissolved in 1M triethylammonium bicarbonate, pH 8.4–8.6 (Fluka) with 20 mg of peptide in 1 ml dimethylsuphoxide and stirring at room temperature for 18 h. Following lyophilization of the solution, the conjugated peptide was solubilized in H20 and purified by RP-HPLC as previously described using a gradient of 0% to 40% Buffer B over 40 min. The peak fractions were analyzed by LC–MS on an Agilent 1100 LC-MSD. The calculated molecular weights of the peptide were in agreement with the mass found.Solid phase synthesis of peptides was carried out on an Intavis peptide synthesizer, starting with 50 μmole Rink Amide AM low loading resin, using N-Fmoc protected amino acids and HCTU as the coupling reagent . At the site of lysine modification Fmoc-Lys(ivDde)-OH was incorporated and the N terminus of the peptides was acetylated. Following chain assembly, the ivDde group was removed by three treatments of the peptidyl-resin with 15 ml of 2% hydrazine/dimethylformamide (DMF) for 1 h at RT and subsequent washing with DMF and dichloromethane (DCM). Next the peptidyl-resin was reacted with 8.3 equivalents (eq) of R-2-bromopropionic acid and 8.3 eq of diisopropylcarbodiimide in DMF for 2 h at room temperature, followed by washing with DMF and DCM. Cleavage and deprotection of the peptide was achieved by adding the peptidyl-resin to 10 ml of 92.5% trifluoroacetic acid (TFA), 2.5% ethanedithiol, 2.5% triisopropyl silane, and 2.5% H2, 10% glycerol, 27% 2-propanol and 0.09 M HEPES pH 7.5 and were harvested in 25% ethylene glycol. Peptide K105-CoA with xEco2 crystallized in 40% ethylene glycol, 0.1 M HEPES pH 7.5, 5% PEG 3K. Data for all crystal forms was collected at Diamond light source on beamline I02 or I03. For crystal of K106-CoA molecular replacement was carried out in PhaserxEco2 ACT was concentrated to 15 mg/ml and crystallized alone or with the addition of the conjugated peptides K106-CoA or K105-CoA. The peptide-free form of xEco2 crystallized in 0.2 M NaCl, 0.1 M imidazole pH 7 and 0.864 M ammonium phosphate. Crystals were harvested with the addition of 30% ethylene glycol. K106-CoA and xEco2 crystallized in 0.18 M MgClin vivo acetylation of Smc3 by mutant Eco1, cells were grown in YNB CSM-MET and arrested in G1 by addition of alpha factor 1 H. Prior to release, cells were transferred to YPD + IAA + alpha factor. Upon G1 arrest, cells were released into nocodazole for 90 minutes, when proteins were TCA extracted. 15 μg of proteins were separated on 10% SDS-PAGE, transferred and submitted to Western blotting with anti-Acetyl Smc3 (K. Shirahige) and anti HA .For the spot assays pMET-eco1AID (Y4766) cells were transformed with an empty URA3 plasmid or a URA3 plasmid carrying different versions of Eco1 expressed under Eco1 endogenous promoter. Serial dilutions of stationary cultures were dropped onto YNB-MET-URA as well as YPD + IAA plates and grown for 2 days at 30 °C. To determine the Accession codes: Atomic coordinates and structure Fsu factors of xEco2, xEco2-K105-CoA, and xEco2-K106-CoA have been deposited in the Protein Data Bank under accession codes 5N1U, 5N1W, and 5N22 respectively.How to cite this article: Chao, W. C. H. et al. Structural Basis of Eco1-Mediated Cohesin Acetylation. Sci. Rep.7, 44313; doi: 10.1038/srep44313 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.