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Articles| January 21 2003
HA95 and LAP2β mediate a novel chromatin–nuclear envelope interaction implicated in initiation of DNA replication
Sandra Martins,
Sandra Martins
1Institute of Medical Biochemistry, University of Oslo, Oslo 0317, Norway
Sissel Eikvar,
Sissel Eikvar
Kazuhiro Furukawa,
Kazuhiro Furukawa
2Department of Chemistry, Faculty of Science, Niigata University, Niigata 950-2181, Japan
Philippe Collas
Address correspondence to Philippe Collas, Institute of Medical Biochemistry, P.O. Box 1112 Blindern, University of Oslo, Oslo 0317, Norway. Tel.: 47-2285-1066. Fax: 47-2285-1058. E-mail: philippe.collas@basalmed.uio.no
Received: October 07 2002
Revision Received: December 09 2002
Accepted: December 09 2002
Sandra Martins, Sissel Eikvar, Kazuhiro Furukawa, Philippe Collas; HA95 and LAP2β mediate a novel chromatin–nuclear envelope interaction implicated in initiation of DNA replication . J Cell Biol 20 January 2003; 160 (2): 177–188. doi: https://doi.org/10.1083/jcb.200210026
HA95 is a chromatin-associated protein that interfaces the nuclear envelope (NE) and chromatin. We report an interaction between HA95 and the inner nuclear membrane protein lamina-associated polypeptide (LAP) 2β, and a role of this association in initiation of DNA replication. Precipitation of GST–LAP2β fusion proteins and overlays of immobilized HA95 indicate that a first HA95-binding region lies within amino acids 137–242 of LAP2β. A second domain sufficient to bind HA95 colocalizes with the lamin B–binding domain of LAP2β at residues 299–373. HA95–LAP2β interaction is not required for NE formation. However, disruption of the association of HA95 with the NH2-terminal HA95-binding domain of LAP2β abolishes the initiation, but not elongation, of DNA replication in purified G1 phase nuclei incubated in S-phase extract. Inhibition of replication initiation correlates with proteasome-mediated proteolysis of Cdc6, a component of the prereplication complex. Rescue of Cdc6 degradation with proteasome inhibitors restores replication. We propose that an interaction of LAP2β, or LAP2 proteins, with HA95 is involved in the control of initiation of DNA replication.
The nuclear envelope (NE)* mediates key nuclear functions by interacting with chromatin. The inner nuclear membrane (INM) harbors integral proteins that interact with the nuclear lamina and chromosomes. Among these, lamina-associated polypeptide (LAP) 2β binds chromatin in a phosphorylation-dependent manner (Foisner and Gerace, 1993; Furukawa et al., 1995) and contains a LAP2, emerin, MAN-1 (LEM) domain (Lin et al., 2000) that interacts with the DNA-bridging protein, barrier-to-autointegration factor (BAF) (Furukawa, 1999). At least six LAP2 isoforms are produced from alternative mRNA splicing in mammals (Berger et al., 1996). LAP2 proteins share an NH2-terminal 187–amino acid domain that binds DNA (Cai et al., 2001) and contains the LEM motif (residues 111–152) (Furukawa, 1999; Shumaker et al., 2001). LAP2β also interacts with the germ cell less (GCL) transcription regulator through residues 219–328 and mediates transcriptional repression alone or together with GCL (Nili et al., 2001). A lamin B–binding domain specific for LAP2β has been identified at residues 299–373 (Furukawa and Kondo, 1998). Injection of LAP2β fragments in living cells (Yang et al., 1997) and in vitro studies (Gant et al., 1999) suggests that LAP2β is involved in nuclear assembly.
We, and others, have cloned a 95-kD nuclear protein named HA95/NAKAP95/HAP95 (Orstavik et al., 2000; Seki et al., 2000; Westberg et al., 2000). HA95 displays partial homology to AKAP95, a member of the A-kinase anchoring protein family, but it lacks a protein kinase A (PKA)–binding domain. HA95 appears in photobleaching experiments as a stable protein and cofractionates with chromatin and a nuclease- and salt-resistant "matrix." HA95 associates with itself (Orstavik et al., 2000) and interacts with RNA helicase A (RHA) to enhance expression of a constitutive transport element (CTE) involved in nuclear export of retroviral RNA (Westberg et al., 2000; Yang et al., 2001). In vitro nuclear breakdown and reassembly assays combined with antibody-blocking experiments have shown that HA95 is involved in NE breakdown and chromatin condensation, whereas a role in nuclear membrane reassembly is unlikely (Martins et al., 2000).
Initiation of DNA replication involves the assembly of prereplication complexes (preRCs) at origins of replication in G1 (Kelly and Brown, 2000; Bell and Dutta, 2002). PreRCs include the origin recognition complex (ORC), the minichromosome maintenance (MCM) complex, and the monomeric Cdc6 protein (Bell and Dutta, 2002). ORC recruits Cdc6 into the preRC in G1, and Cdc6 in turn promotes loading of MCM proteins on chromatin (Bell and Dutta, 2002). In human cells, Cdc6 levels are relatively stable during interphase (Williams et al., 1997; Saha et al., 1998). Nonetheless, a fraction of Cdc6 is exported out of the nucleus (Coleman et al., 1996) while another remains associated with chromatin during S and G2 phases (Coverley et al., 2000; Mendez and Stillman, 2000). Proteolysis of free Cdc6, not assembled into preRCs, has also been reported (Coverley et al., 2000). Thus, after origin firing at the start of S phase, preRCs are dissociated, ensuring a single round of replication per cell cycle.
We provide evidence here that HA95 interacts with LAP2β via two distinct domains. Blocking the association of HA95 with LAP2β does not affect nuclear assembly in vitro. However, disruption of the association of HA95 with the NH2-terminal HA95-binding domain of LAP2β triggers proteasome-mediated degradation of Cdc6 and inhibits initiation, but not elongation, of replication. The results suggest an implication of the association of HA95 with LAP2β in regulating the initiation phase of DNA replication.
HA95 and LAP2β coprecipitate in interphase
HA95 coprecipitates with a protein complex containing LAP2β from the transformed human B cell line Bjab in interphase (Martins et al., 2000). We now show that in Bjab cells, HA95 and LAP2β coprecipitated in interphase but not at mitosis, regardless of the precipitating antibody (Fig. 1 A). Immunoblots of the nonprecipitated (S) fractions indicate that in interphase, HA95 coprecipitated all detectable LAP2β, whereas LAP2β did not precipitate all HA95 (Fig. 1 A). Immunoprecipitation of an HA95–Myc fusion protein stably expressed in Bjab cells using an anti-Myc antibody also coprecipitated LAP2β in interphase but not at mitosis (Fig. 1 B). These results suggest a cell cycle–dependent interaction of HA95 with LAP2β; however, not all HA95 resides in a complex with LAP2β.
The strength of interaction between HA95 and LAP2β within the complex was evaluated by a 30-min extraction of anti-HA95 immune precipitates (HA95-IPs) with 0–1.25 M NaCl. Fig. 1 C shows that the interaction resisted a 0.75 M NaCl wash but was completely disrupted with 1.25 M NaCl. High salt resistance of the interaction suggests a strong association between HA95 and LAP2β, confirming our earlier cross-linking data (Martins et al., 2000).
Mapping of the HA95-binding domains of LAP2β
To map the domains of LAP2β involved in the interaction with HA95, GST–LAP2β fusion polypeptides were produced (Fig. 2 A) (Furukawa et al., 1995, 1997, 1998). Binding of each peptide to HA95–Myc was determined in GST precipitations after incubation of the peptides in a nuclear extract from Bjab cells expressing HA95–Myc. Control extracts were incubated with GST or glutathione beads alone. GST precipitates were immunoblotted using anti-Myc antibodies. Fig. 2 B shows that LAP2β(1–452), (1–397), (1–298), (137–373), (137–298), (243–397), (243–373), (299–397), and (299–373) precipitated HA95–Myc. In contrast, LAP2β(1–193), (1–85), or (194–298) did not precipitate HA95–Myc. Thus, a first HA95-binding domain localizes to amino acids 137–242 of LAP2β and a second domain coincides with the lamin B–binding domain at residues 299–373. We designated these domains HA95-NBD (for HA95 NH2-terminal binding domain) and HA95-CBD (HA95 COOH-terminal binding domain), respectively.
The Myc tag itself did not bind GST or LAP2β. LAP2β(137–373), which binds HA95–Myc, did not coprecipitate a stably expressed Myc-tagged p70S6 kinase (Fig. 2 C). Furthermore, anti-Myc antibodies coprecipitated LAP2β (1–397) but not the nonbinding fragment LAP2β(1–193) from a nuclear extract of Bjab cells expressing HA95–Myc (unpublished data). Altogether, the results indicate that HA95 interacts with recombinant LAP2β.
Direct association of LA2β with HA95 was demonstrated in an overlay assay. HA95-IP proteins were resolved by SDS-PAGE, blotted, and overlaid with 50 μM of each GST–LAP2β fragment. The same GST–LAP2β peptides found to precipitate HA95 also bound HA95 in the overlay, as detected with anti-GST antibodies (Fig. 2 D, top). Moreover, all LAP2β fragments containing the lamin B–binding domain (residues 299–373) bound to immunoprecipitated and immobilized lamin B (Fig. 2 D, bottom). However, two of the HA95-binding peptides, LAP2β(1–298) and LAP2β(137–298), did not bind lamin B, confirming the existence of an HA95-binding domain (HA95-NBD) distinct from the lamin B–binding region.
To determine whether HA95-binding LAP2β peptides would disrupt endogenous HA95–LAP2β association, dissociation experiments were performed using GST–LAP2β peptides harboring both HA95-binding domains (LAP2β [137–373]) or either HA95-NBD (LAP2β[137–298]) or HA95-CBD (LAP2β[299–373]). HA95-IPs were incubated for 1 h with 100 μM GST–LAP2β peptides, HA95-IPs were sedimented, and dissociation of LAP2β from HA95 was monitored by immunoblotting. LAP2β(137–373) completely dissociated LAP2β from HA95-IP (Fig. 2 E, lanes 1 and 2), however peptides containing HA95-NBD or HA95-CBD were ineffective (lanes 3–6). Nevertheless, two sequential 30-min incubations of HA95-IPs with LAP2β(137–298) followed by LAP2β(299–373) led to dissociation of the complex (lanes 7 and 8), and reversing the order of peptide addition produced similar results (lanes 9 and 10). We concluded that disruption of both HA95-binding domains was required for dissociation of LAP2β from HA95 in vitro. The data also argue that LAP2β(137–298) and LAP2β(299–373) peptides are capable of dissociating HA95 from HA95-NBD and HA95-CBD, respectively.
Inhibition of LAP2β binding to HA95 does not affect nuclear reassembly in vitro
To determine whether interaction between HA95 and LAP2β was involved in NE assembly, we assessed the ability of HA95-binding GST–LAP2β peptides to compete with LAP2β for membrane targeting to chromosomes. Purified HeLa nuclei were disassembled in mitotic extract. The resulting condensed chromosomes contained HA95 and BAF, but no A- or B-type lamins, LAP2β, or lamin B receptor (LBR) (Fig. 3 A). After preincubation with 10 μM of each GST–LAP2β peptide, chromosomes were sedimented and peptide binding was examined by immunofluorescence using anti-GST antibodies. All peptides except GST–LAP2β(194–298) bound chromatin (Fig. 3 B), as anticipated from their ability to bind HA95 (Fig. 2), DNA, or chromatin (see Introduction). Chromosomes were resuspended in interphase extract (Fig. 3 C, Input chrom.) under conditions promoting nuclear assembly. Nuclear morphology was examined by phase contrast microscopy and membrane labeling with DiOC6 after 2 h (Fig. 3 C). Without peptide or with GST alone, >80% of chromatin masses supported nuclear reformation. In contrast, LAP2β fragments (1–452), (1–397), (137–373), (243–397), (299–397), (243–373), and (299–373) inhibited nuclear assembly. Each of these peptides contained the lamin B–binding domain/HA95-CBD. Peptides that do not bind HA95 (nor lamin B) (LAP2β[1–193], [1–85], and [194–298]) did not block membrane assembly, neither did LAP2β(1–298) or (137–298), which both contain HA95-NBD.
To address whether disruption of the of HA95–LAP2β interaction altered global nuclear organization, localization of LAP2β, A- and B-type lamins, and BAF was analyzed by immunofluorescence in nuclei reconstituted in the presence of either no peptide, a peptide not binding to HA95 (LAP2β[1–193]), or peptides containing both HA95-NBD and HA95-CBD (LAP2β[137–373]), HA95-NBD only (LAP2β[137–298]), or HA95-CBD only (LAP2β[299–373]). Anti-GST immunolabeling shows peptide distribution in the resulting nuclei or chromatin masses (Fig. 3 D). Distribution of LAP2β, lamins A/C and B, or BAF was not distinctively altered in nuclei reconstituted with HA95-binding or nonbinding LAP2β peptides (Fig. 3 D). LAP2β targeting to chromatin and nuclear assembly of A- and B-type lamins, or lack thereof, were verified by immunoblotting nuclei or chromatin purified from the extract (Fig. 3 E). The blots also showed that levels of chromatin-associated BAF were not significantly altered by GST–LAP2β fragments (Fig. 3 E). We concluded that LAP2β fragments containing the lamin B–binding domain and HA95-CBD prohibit nuclear reformation. However, peptides harboring HA95-NBD only are not inhibitory and do not notably affect the distribution of NE proteins or BAF in the reassembled nuclei. Thus, we could not attribute an effect of HA95 binding per se on NE assembly in vitro.
LAP2β(137–298) inhibits initiation of DNA replication in intact nuclei in vitro
To determine whether interphase nuclear functions were affected by the HA95–LAP2β association, we monitored the effect of disrupting the LAP2β–HA95 interaction on DNA replication in purified G1-phase nuclei. To this end, we adapted an in vitro replication assay from that of Krude et al. (1997).
Nuclei were isolated from G1-phase HeLa cells. GST–LAP2β peptides were introduced into the nuclei after mild treatment with lysolecithin. Lysolecithin was previously shown not to affect dynamic properties of isolated nuclei in in vitro nuclear disassembly assays (Collas et al., 1999). Peptides were taken up by ∼90% of the nuclei, as shown by immunofluorescence using anti-GST antibodies (Fig. 4 A; GST–LAP2β[1–452] is shown). Control and peptide-loaded nuclei were incubated for 3 h in a concentrated (25–30 mg/ml) nuclear and cytosolic extract from S-phase HeLa cells containing [α32P]dCTP, dNTPs, GTP, and an ATP-regenerating system to promote replication. Under these conditions, G1 nuclei loaded with GST–LAP2β(1–452) were capable of importing an exogenous BSA–nuclear localization signal conjugate (unpublished data) or the replication factor Cdc6 (Fig. 4 B). Import was ATP and GTP dependent and blocked by preincubation of the nuclei with antibodies against nucleoporins (Fig. 5 B, mAb414). These results indicate that import took place through nuclear pores rather than passively through a damaged NE, and confirmed a previous report of physiological import of transcription factors by nuclei purified as previously described (Landsverk et al., 2002). We also tested whether peptides containing HA95-NBD or HA95-CBD introduced into G1 nuclei would inhibit nuclear import under the conditions described above, as this would be expected to affect DNA replication. Fig. 4 C shows that none of the peptides distinctly impaired import of Cdc6. Notably, import was permitted by the HA95-NBD–containing peptide LAP2β(137–298) and blocked by mAb414. Lastly, the nuclear DNA did not undergo any detectable degradation upon incubation of the G1 nuclei in the extract at 4°C or 37°C, as judged by TUNEL analysis (Fig. 4 C) and DNA agarose gel electrophoresis (Fig. 4 D). These results indicate that isolated G1 nuclei are functional in import, can be manipulated to introduce peptides, and do not undergo detectable DNA degradation in S-phase extract, and that LAP2β fragments containing either HA95-binding domain do not block nuclear import of Cdc6 in vitro.
Having characterized the nuclei, the effect of GST–LAP2β peptides on replication was examined. Replication was assayed by incorporation of [α32P]dCTP, DNA electrophoresis, and phosphorImaging (Fig. 5 A). Absence of GST–LAP2β peptide or loading GST alone into G1 nuclei did not hinder replication. As expected, DNA synthesis was blocked with 50 μM aphidicolin in the extract. However, LAP2β(1–452) and (1–397) abolished replication and peptides containing HA95-CBD (LAP2β[243–397], [299–397], [243–373], and [299–373]) impaired replication efficiency by ∼50%. Furthermore, LAP2β(1–298), (1–193), (137–373), and (137–298) inhibited replication, whereas LAP2β(194–298) did not. We also ruled out the possibility that the DNA replication signal detected in G1 nuclei represented an elongation phase in already replicating nuclei, because G1 nuclei incubated in extract from G0-arrested cells did not replicate (Fig. 5 B).
Initiation of replication requires the cyclin A–Cdk2 complex (Stoeber et al., 1998). Thus, to provide evidence that DNA synthesis in G1 nuclei was due to true initiation of replication, we showed that DNA synthesis was inhibited with 10 μM of the Cdk2 inhibitor, olomoucine, in the assay (Stoeber et al., 1998) (Fig. 5 B). Similar results were obtained with 500 μg/ml dimethylaminopurine, a nonspecific protein kinase inhibitor (unpublished data). Altogether, the results indicate that peptides containing HA95-CBD partially inhibit replication in G1 nuclei. However, those containing HA95-NDB completely abolish replication. We ruled out an involvement of the GCL-binding domain of LAP2β (residues 219–298) in inhibition of replication by fragment 137–298 because LAP2β(194–298) was not inhibitory.
As described earlier in the nuclear reconstitution experiment, none of the peptides altered the immunofluorescence labeling pattern of LAP2β, A- and B-type lamins, or BAF in the G1 nuclei examined (unpublished data; see also below). In addition, Western blot analysis of the G1 nuclei loaded with the peptides shows that neither peptide affected BAF levels (or HA95) in the nuclei (Fig. 5 C). As LAP2β(1–193) and (137–298) block replication whereas LAP2β(194–298) and (1–85) allow replication (Fig. 5 D), the data suggest that region 137–193 of LAP2β is involved in replication initiation without affecting the distribution of NE proteins or the amount of BAF in the nuclei.
To establish that the LAP2β(137–298) peptide inhibited semiconservative DNA replication in G1 nuclei, BrdU substitution and density gradient centrifugation of 32P-labeled DNA was performed. G1 nuclei loaded with no peptide, GST alone, or LAP2β(137–298) were incubated for 3 h in S-phase extract containing BrdU and [α32P]dCTP to quantitate replication. DNA was analyzed by CsCl gradient centrifugation. In control nuclei, the cytosol promoted DNA synthesis, producing primarily hemisubstituted (heavy-light [HL]) DNA, indicative of semiconservative replication (Fig. 5 E). In contrast, no peak of hemisubstituted DNA was detected in nuclei loaded with LAP2β(137–298), consistent with replication inhibition detected by 32P incorporation. We concluded that LAP2β(137–298) interfered with semiconservative replication in G1 nuclei.
Disrupting LAP2β–HA95 interaction does not affect the elongation phase of DNA replication in vitro
We next determined whether the elongation phase of replication was also affected by LAP2β peptides. Nuclei isolated from S-phase HeLa cells were loaded with each GST–LAP2β peptide (Fig. 5 F, left; GST–LAP2β[1–452] is shown) and incubated in S-phase extract under conditions promoting replication. Remarkably, all peptides supported DNA synthesis to the same extent as a control without peptide (Fig. 5 F). In addition, to validate our DNA replication assay, we showed that DNA synthesis occurred in S-phase nuclei incubated in extract from G0 cells, reflecting the replicating state of these nuclei (Fig. 5 B). In contrast, G0 nuclei did not synthesize DNA in S-phase extract. Note, however, a faint 32P label in G0 nuclei in the S-phase extract, due to a minor proportion of slowly replicating nuclei in the G0 cell population (unpublished data).
HA95 coimmunoprecipitates with the Cdc6 protein in G1 phase
Initiation of DNA replication requires the assembly of preRCs at origins of replication in G1. The chromatin-bound ORC complex recruits Cdc6, which in turn promotes targeting of MCM proteins. HA95 and Cdc6 were found to coimmunoprecipitate from G1-phase HeLa cells; nevertheless, whereas anti-HA95 antibodies precipitated all detectable Cdc6, a substantial fraction of HA95 did not associate with the Cdc6 immune precipitate (Fig. 6 A). In S phase however, Cdc6 and HA95 did not coprecipitate (Fig. 6 A), despite the reported persistence of a fraction of Cdc6 on chromatin beyond G1 (Mendez and Stillman, 2000).
Disruption of the interaction between HA95 and LAP2β via HA95-NBD in G1 induces proteolysis of Cdc6
The role of Cdc6 on preRC assembly led us to investigate the fate of Cdc6 in G1 nuclei after disruption of the HA95–LAP2β association via HA95-NBD or HA95-CBD. GST–LAP2β peptides were introduced into purified G1 HeLa nuclei and nuclei were incubated in nuclear isolation buffer for 1 h. Immunoblotting analyses of the nuclei show that nuclei loaded with GST alone or with LAP2β(299–373) contained Cdc6 (Fig. 6 B, lanes 1–3). However, nuclei loaded with LAP2β(137–298) or LAP2β(1–452) harbored no detectable Cdc6 (lanes 4 and 5). Additionally, blots of the whole incubation mix (nuclei and buffer) revealed no Cdc6 either, indicating that Cdc6 was degraded under these conditions (lane 9; see below). Note that p53 was also degraded in nuclei containing peptides harboring HA95-NBD, however, Orc2, a component of the preRC, was not affected (lanes 4 and 5).
The ORC large subunit has recently been shown to be degraded by ubiquitin-mediated proteolysis in human cells (Mendez et al., 2002). Thus degradation of Cdc6 by the 25S proteasome was examined. Inhibition of the proteasome by incubation of nuclei containing LAP2β(137–298) with 25 μM of the proteasome inhibitors LLnL or β-lactone during peptide loading and incubation in buffer prevented degradation of Cdc6 (Fig. 6 B, lanes 6 and 7). However, the calpain inhibitor LLM, used as a control, did not block Cdc6 proteolysis (lane 8). Inhibition of degradation of p53, a known substrate of ubiquitin-mediated proteolysis, with LLnL or β-lactone confirmed the efficacy of the inhibitors (Fig. 6 B). Introduction of LAP2β(137–298) in S-phase nuclei did not elicit degradation of Cdc6 (or p53 or Orc2; unpublished data). These results suggest that dissociation of HA95 from HA95-NBD in G1- but not S-phase nuclei triggers degradation of Cdc6 by the proteasome. Because p53 is also proteolyzed by LAP2β(137–298), the data also suggest that the peptide promotes degradation of a subset of nuclear proteins.
As G1 nuclei containing LAP2β(137–298) do not replicate DNA in S-phase extract, we determined whether proteasome inhibitors would rescue S-phase entry of these nuclei. G1 nuclei were loaded with LAP2β(137–298) in the presence of LLnL or β-lactone and incubated for 3 h in S-phase extract containing [α32P]dCTP under conditions promoting replication. Fig. 6 C shows that LLnL and β-lactone relieved the inhibition of DNA replication imposed by LAP2β(137–298). The calpain inhibitor LLM, however, had no effect and, as expected, LLnL or β-lactone did not affect replication of LAP2β(299–373)-loaded G1 nuclei (Fig. 6 C). Therefore, inhibition of the proteasome enables entry of the G1 nuclei into a replication phase.
Injection of LAP2β(137–298) into G1 nuclei inhibits entry into S phase in vivo
The significance of HA95 interaction with HA95-NBD or HA95-CBD was further investigated in vivo by injections of ∼5 nM GST–LAP2β peptides into the nuclei of HeLa cells in early G1 (2 h after release from mitotic arrest). Injections were verified by nuclear retention of a 150-kD FITC–dextran (see below and Fig. 8 A).
We first assessed whether the distribution of INM and lamina proteins was altered in the injected nuclei. As seen earlier in in vitro–reconstituted nuclei, GST–LAP2β peptides were detected throughout the nucleus for the most part, with a propensity of the anti-GST antibody to decorate the nuclear periphery more strongly (Fig. 7, GST). This, however, was not specific for the peptide injected (Fig. 7, bottom three rows). Immunofluorescence analysis of peptide- and mock (buffer)-injected cells indicated that LAP2β and B-type lamins remained localized at the NE 2–3 h after injection with either peptide (Fig. 7). Similar results were obtained for LBR, emerin, and A-type lamins (unpublished data). Additionally, no alteration in the localization of BAF in peptide-injected and control cells was detected (Fig. 7). BAF remained distributed throughout the nucleoplasm with an enrichment around the periphery. Thus, we could not attribute a noticeable effect of intranuclear peptide injection in G1 on overall nuclear architecture.
To examine the effect of peptide injection in G1 on DNA replication, injected G1-phase cells were cultured with 10 μM BrdU for 10 h and DNA synthesis was monitored using anti-BrdU antibodies. Fig. 8 (A and B) shows that 95% of mock-injected cells underwent DNA synthesis, which was inhibited by 50 μM aphidicolin. Likewise, cells injected with LAP2β(1–85) or GST alone replicated DNA. However, LAP2β(137–298) abolished DNA synthesis in 90% of the cells, whereas LAP2β(299–373) had no effect. Injection of LAP2β(137–298) in S-phase nuclei was not inhibitory (unpublished data), indicating that once DNA synthesis is initiated, disruption of the LAP2β–HA95 interaction via HA95-NBD has no effect.
Additional immunofluorescence analysis of injected G1 HeLa cells indicated that Cdc6 was undetectable in LAP2β (137–298)-injected cells, whereas intranuclear labeling was evident in noninjected cells (Fig. 8 C, arrow) or in cells injected with GST alone or LAP2β(299–373) (Fig. 8 C). Note that Orc2 was not degraded in LAP2β(137–298)-injected cells, as shown by intranuclear immunolabeling (Fig. 8 C). The results indicate that, as shown biochemically in vitro, LAP2β(137–298) inhibits S-phase entry in vivo. Inhibition correlates with the degradation of Cdc6, but is not due to a displacement of NE proteins or a change in BAF distribution during G1.
Anchoring of the INM to chromatin
This study provides evidence for a novel direct interaction of the NE with the genome, via the INM protein LAP2β and the chromatin- and nuclear matrix–associated protein HA95. The nucleoplasmic 410 amino acids of LAP2β are prompt to interactions with multiple intranuclear ligands (Fig. 9). The first 50 residues are common to all LAP2 proteins and bind DNA (Cai et al., 2001), in consistency with the chromatin-binding property of this region, which causes post-mitotic association of LAP2α with chromosomes (Vlcek et al., 1999). LAP2 proteins also bind the DNA-bridging protein BAF through residues 67–137 (Furukawa, 1999; Shumaker et al., 2001), which include most of the LEM domain (Fig. 9). The significance of this interaction in interphase remains unclear, although a role in chromatin decondensation and nuclear expansion in vitro has been proposed (Segura-Totten et al., 2002). LAP2β also binds GCL, a transcription regulator that interacts with components of the E2F transcription machinery (Nili et al., 2001). LAP2β is capable of reducing the transcription activity of the E2F complex alone or with GCL (Nili et al., 2001), suggesting an involvement of the NE in transcription regulation. It will be interesting to determine whether mutations in NE proteins that cause disease (Vigouroux and Bonne, 2002) also lead to defects in transcription regulation in affected tissues.
We now show that LAP2β also directly interacts with the chromatin-associated protein HA95 via two HA95-binding domains that are distinct from the chromosome-binding region. The NH2-terminal domain (HA95-NBD) localizes within amino acids 137–242, while the COOH-terminal domain (HA95-CBD) is restricted to residues 299–373 (Fig. 9). HA95-NBD partially overlaps with the 187 NH2-terminal amino acids common to all LAP2 proteins, thus HA95-NBD may not be restricted to NE-associated LAP2β and may also exist in intranuclear LAP2 proteins. HA95-CBD coincides with the lamin B–binding domain of LAP2β and is unique to LAP2β, nevertheless LAP2β binds HA95 independently of lamin B in vitro. The functional implications of each HA95-binding domain of LAP2β are important, as disruption of only one or the other has distinct effects on NE assembly or DNA replication (see below). As the binding domains to each ligand are distinct, interaction of LAP2β with either domain may not be exclusive, and it is tempting to speculate that a single protein of the INM may regulate chromatin function at multiple levels in interphase.
Interaction between HA95 and LAP2β is dispensable for nuclear membrane assembly
We previously reported that anti-HA95 antibody-mediated blocking of HA95 function on condensed chromosomes had no inhibitory effect on nuclear membrane assembly in vitro (Martins et al., 2000). This suggested that HA95 may not be involved in membrane targeting to chromatin. Our results indicate that any LAP2β fragment that can bind lamin B inhibits nuclear assembly, however peptides that bind HA95, but not lamin B, fail to inhibit assembly. Thus, competition for LAP2β binding to HA95 has no detectable effect on nuclear formation. Although our results show that the lamin B–binding domain of LAP2β is involved in NE formation (Yang et al., 1997; Gant et al., 1999), they document a lack of effect of HA95 binding activity on nuclear assembly.
Role of association of HA95 with the NH2-terminal HA95-binding domain of LAP2β in DNA replication
Our results suggest that the HA95–LAP2β interaction via HA95-NBD may represent an additional form of control for replication initiation, at least in transformed cells. We ruled out the possibility that disruption of the HA95–LAP2β interaction upon intranuclear introduction of LAP2β fragments affected overall nuclear organization, which in itself might compromise DNA replication (Gant et al., 1999; Moir et al., 2000). Notably, the localization of endogenous LAP2β, A- and B-type lamins, and BAF does not seem to be perturbed by disruption of the interaction of HA95 with HA95-NBD upon nuclear reassembly in vitro, in isolated G1 nuclei, or in G1-phase nuclei in vivo. Additionally, BAF levels remain unaffected in G1 nuclei, arguing that defects in replication induced by LAP2β disruptor peptides do not result from changes in BAF protein levels (Segura-Totten et al., 2002). The HA95-NBD of LAP2(β) partially overlaps with the LEM domain, which interacts with BAF. BAF distribution and levels are not altered by LAP2β peptides containing HA95-NBD, and regions nonoverlapping with the HA95-binding domain were sufficient for BAF binding in yeast two-hybrid assays (Furukawa, 1999). Nonetheless, whether manipulating the association of HA95 with LAP2β interferes with binding of LAP2 proteins to BAF remains to be examined (Shumaker et al., 2001).
How does the HA95–LAP2β interaction affect DNA replication? First, we should emphasize that the domain of HA95-NBD involved in replication (residues 137–193; Fig. 9) is almost entirely included in the 187 amino acids (1–187) common to all LAP2 proteins. Thus, the HA95–LAP2 interaction is probably not restricted to the NE. We propose a hypothesis whereby interaction of HA95 with the NH2-terminal domain of LAP2 proteins and with components of the preRC brings components of the preRC to replication origins, maintains integrity of the preRC, and/or protects preRC components from degradation. It is conceivable that destabilizing the HA95–LAP2β interaction in G1 may displace preRC components, triggering their proteasome-mediated degradation and, as a result, blocking replication initiation. These alternatives are compatible with the distribution of preRCs throughout the genome and the intranuclear localization of LAP2α, a nonmembrane-bound LAP2 isoform (Vlcek et al., 1999). Disruption of the interaction specifically abolishes S-phase entry but has no effect on the elongation phase of replication. Furthermore, HA95 and Cdc6 coimmunoprecipitate in G1 but not S phase, suggesting that HA95 (in)directly interacts with the preRC. The lack of interaction of HA95 with Cdc6 in S phase is consistent with nuclear export or degradation of a fraction of Cdc6 in mammalian cells (Saha et al., 1998; Coverley et al., 2000) and with disassembly of the preRC.
Disruption of HA95 interaction with HA95-NBD in G1 triggers proteasome-mediated degradation of Cdc6 (and p53), whereas proteasome inhibitors block LAP2β(137–298)-induced proteolysis and rescue the replication inhibitory effect of the LAP2β peptide in G1 nuclei. Yet, the inhibitors obviously did not rescue the LAP2β–HA95 interaction, indicating that this association is dispensable for replication. It is likely that the LAP2β(137–298) peptide causes degradation of a class of nuclear proteins, because p53 reacts similarly to Cdc6. This could result from a pleiotropic effect of the peptide, perhaps disturbing other proteins that interact through a similar domain. In any event, inhibition of DNA synthesis by LAP2β(137–298) may be indirect, for example by activating a checkpoint pathway. This speculative hypothesis remains to be tested.
Our results extend the notion that the NE contributes to regulating replication. In Xenopus, disruption of lamina organization alters the distribution of replication factors and inhibits the elongation phase of DNA synthesis (Moir et al., 2000). Thus, replication may be regulated at several levels by nuclear lamins and nuclear membrane–chromatin interactions, in addition to interactions between chromatin-associated proteins.
Multiple functions of HA95 dictated by its interaction with multiple ligands
By interacting with several intranuclear ligands, HA95 emerges as a multifunctional molecule. In addition to its involvement in replication, HA95 binds RHA (Westberg et al., 2000), suggestive of a role in transcription regulation. RHA binds to a CTE involved in nuclear export of unspliced viral RNA, and association of HA95 with RHA enhances CTE-mediated gene expression and promotes nuclear export of unspliced mRNA (Yang et al., 2001). By binding to LAP2β, HA95 may favor viral RNA export by tethering it near the NE.
HA95 also interacts with the catalytic subunit of PKA within the nucleus and localizes the catalytic subunit to sites where it can modulate transcription from specific promoters (Han et al., 2002). Thus, HA95 may act as a targeting molecule for the PKA catalytic subunit in the nucleus. Along this line, it is tempting to speculate that HA95–LAP2β, or HA95–LAP2, interactions may promote targeting or stabilization of signaling molecules in the vicinity of transcription sites and at origins of replication at discrete stages of the cell cycle.
Antibodies and peptides
Anti-Myc antibodies were from Invitrogen. Anti-GST, anti-Cdc6 (H-304 and clone 180.2), anti-Orc2, goat anti–lamin B antibodies, and anti-p53 antibodies were from Santa Cruz Biotechnology, Inc. Anti-BrdU antibodies were from Sigma-Aldrich. Affinity-purified polyclonal antibodies against LBR, LAP2β, A- and B-type lamins (gifts from J.-C. Courvalin, Institut J. Monod, Paris, France), BAF (a gift from K. Wilson, Johns Hopkins University School of Medicine, Baltimore, MD), and HA95 were described elsewhere (Buendia and Courvalin, 1997; Orstavik et al., 2000; Haraguchi et al., 2001). LLnL, LLM, and β-lactone were from Calbiochem. GST–LAP2β peptides were expressed as previously described (Furukawa et al., 1995, 1997, 1998).
Cells and nuclei
Bjab cells were grown in RPMI 1640/10% FCS (GIBCO BRL) (Orstavik et al., 2000). HeLa cells were cultured in EMEM/10% FCS (GIBCO BRL). HeLa cells were synchronized in M phase with 1 μM nocodazole for 18 h. To allow cell cycle reentry, cells were plated at 2.5 × 106 cells in 162-cm2 flasks. G1- and S-phase cells were harvested (or microinjected) 2 and 12 h, respectively, after release from mitotic arrest. Cells were arrested in G0 by a 5-d culture under confluent conditions without serum. Nuclei were isolated from Bjab cells and from G0-, G1-, or S-phase HeLa cells by Dounce homogenization (Martins et al., 2000). For nuclear reassembly assays, nuclei were isolated from confluent HeLa cells and used fresh of frozen/thawed (Steen et al., 2000). Freshly isolated nuclei were used in replication assays.
Microinjection and BrdU incorporation
HeLa cells released from M-phase arrest were seeded on coverslips. Within 2 h, nuclei were microinjected with 25 pl PBS containing 10 μg/ml 150-kD FITC–dextran to visualize injections, and ∼5 nM indicated GST–LAP2β peptide. Cells were cultured in EMEM/10% FCS for up to 10 h without or with 100 μM BrdU and processed for immunofluorescence or BrdU incorporation analysis. S-phase cells were injected 10–12 h after release from M-phase arrest. Approximately 50 cells were injected per treatment in two to three replicates. To detect BrdU incorporation, cells were fixed with methanol for 5 min, blocked, and overlaid with anti-BrdU antibodies (1:200 dilution) and TRITC-conjugated anti-BrdU antibodies.
Immunological procedures
Immunoblotting analysis was performed as previously described (Martins et al., 2000) using antibodies against HA95 (1:250 dilution), Myc (1:1,000), GST (1:1,000), B-type lamins (1:1,000), lamin A/C (1:500), LAP2β (1:500), LBR (1:500), BAF (1:500), p53 (1:500), Cdc6 (1:500), and Orc2 (1:500). For immunoprecipitations, cells or nuclei were sonicated in IP buffer (10 mM Hepes, pH 7.5, 10 mM KCl, 2 mM EDTA, 1% Triton X-100, 1 mM DTT, and protease inhibitors) and lysates were centrifuged at 15,000 g for 15 min. Immunoprecipitations were performed from the supernatants with relevant antibodies (1:50 dilutions) as described earlier (Martins et al., 2000).
GST precipitation
Nuclei isolated from Bjab cells (109 nuclei/ml) were sonicated in GST precipitation buffer (300 mM KCl, 20 mM Hepes, pH 7.6, 0.1% Triton X-100, 1 mM DTT, 5 mM benzamidine, and protease inhibitors) and the lysate was centrifuged at 10,000 g. The supernatant (nuclear extract) was incubated with specified GST–LAP2β peptides (1 or 5 μg/μl) overnight at 4°C with rotation. GST precipitations were performed using glutathione beads coated with 10 mg/ml BSA and pellets were washed in GST precipitation buffer. Proteins were eluted in SDS sample buffer.
Overlay assays
HA95-IPs proteins resolved by 10% SDS-PAGE were blotted onto nitrocellulose, blocked with 5% milk in Tris-buffered saline/0.01% Tween 20 (TBST) for 1 h, washed in TBST, and overlaid with 10 μM GST–LAP2β peptides for 2 h in TBST. Membranes were washed in TBST and peptide binding was detected using anti-GST antibodies and peroxidase-conjugated secondary antibodies.
Nuclear reconstitution assay
Condensed, membrane-free chromatin masses were prepared from HeLa nuclei disassembled in mitotic extract (Martins et al., 2000). After sedimentation through 1 M sucrose, chromosomes were resuspended in peptide-binding buffer (100 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 20 mM Hepes, pH 7.5, 1 mM DTT, and protease inhibitors) containing 5 μM of indicated GST–LAP2β peptides.
After 1 h at room temperature, chromosomes were sedimented, washed, and either immunologically analyzed or resuspended in nuclear reassembly extract. In this case, chromosomes (2 μl) were resuspended in 40 μl of an interphase HeLa cell 200,000 g cytosolic extract at 5,000 chromatin masses/μl, containing 4 μl mitotic membranes, an ATP-regenerating system (1.2 μl), and 100 μM GTP (0.4 μl) (Steen et al., 2000). After 2 h at 30°C, nuclear assembly was examined by phase contrast microscopy, membrane staining with 10 μg/ml DiOC6, or by immunofluorescence. Nuclei or chromatin masses were also sedimented through 1 M sucrose, washed, and solubilized in SDS sample buffer.
Loading of nuclei with GST–LAP2β peptides
Peptide loading into isolated HeLa nuclei was performed as described earlier (Collas et al., 1999). In brief, nuclei were mildly permeabilized with lysolecithin and incubated for 1 h with indicated GST–LAP2β peptides (100 μM) or GST alone. Nuclei were washed by sedimentation through 1 M sucrose and held on ice until use. Lysolecithin-treated nuclei were capable of active protein import in vitro (see Results).
In vitro replication and quantification of DNA synthesis
Replication was assayed by incorporation of [α32P]dCTP into newly synthesized DNA. Isolated G0-, G1-, or S-phase nuclei (preloaded with GST–LAP2β peptides, as indicated) were incubated for 3 h at 5,000 nuclei/μl in 40 μl of S-phase extract (unless indicated otherwise) containing the ATP-regenerating system, 100 μM GTP, a buffered mix of dNTPs (40 mM Hepes, pH 7.8, 7 mM MgCl2, 0.1 mM each of dATP, dGTP, dTTP, and dCTP; 2 μl) and 1 μl [α32P]dCTP (3,000 Ci/mmol; Amersham Biosciences). Concentrated S-phase whole cell extracts were prepared from S-phase HeLa cells collected 15 h after release from mitotic arrest. Cells were lysed by Dounce homogenization in cell lysis buffer (Martins et al., 2000), and then briefly sonicated on ice to lyse nuclei and release soluble nuclear components. The lysate was sedimented at 15,000 g for 15 min and then at 200,000 g for 2 h, both at 4°C. Protein concentration of the extract was 25–30 mg/ml.
At the end of incubation in extract, samples were mixed with 1 volume of 20 mM Tris (pH 7.5) and 1 mg/ml proteinase K and digested for 2 h at 37°C. Samples were mixed by pipetting and 5-μl aliquots were electrophoresed through 0.8% agarose. Gel loading was assessed by ethidium bromide staining. Samples contained equal numbers of nuclei and sedimentation steps were eliminated to avoid loss of nuclei (Gant et al., 1999). Signals were quantified by phosphorImaging or by autoradiography.
BrdU density substitution
Density substitutions were done as previously described (Gant et al., 1999) in reactions consisting of isolated G1 nuclei in S-phase extract containing [α32P]dCTP and 0.5 mM BrdU. Samples were digested with proteinase K and DNA was extracted with phenol–chloroform then chloroform, ethanol precipitated, and dissolved in 100 μl Tris-EDTA buffer. Samples were mixed with 12 ml of 1.75 g/ml CsCl and centrifuged for 45 h at 60,000 g. 40 fractions were collected, an aliquot was counted by liquid scintillation, and the refractive index of each fraction was measured (Gant et al., 1999).
We are grateful to Drs. K. Wilson and J.-C. Courvalin for antibodies.
This work was supported by the Portuguese Foundation for Science and Technology (S. Martins), the Research Council of Norway, the Norwegian Cancer Society, and the Human Frontiers Science Program (P. Collas).
Abbreviations used in this paper: BAF, barrier-to-autointegration factor; CBD, COOH-terminal binding domain; CTE, constitutive transport element; GCL, germ cell less; HA95-IP, anti-HA95 immune precipitate; INM, inner nuclear membrane; LBR, lamin B receptor; LEM, LAP2, emerin, MAN-1; MCM, minichromosome maintenance; NBD, NH2-terminal binding domain; NE, nuclear envelope; ORC, origin recognition complex; PKA, protein kinase A; preRC, prereplication complex; RHA, RNA helicase A.
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HA95 coprecipitates with LAP2β in interphase. (A) HA95 or LAP2β was immunoprecipitated (IP) from interphase and mitotic Bjab cells. Precipitates (P) and supernatants (S) were immunoblotted using anti-LAP2β or anti-HA95 antibodies. (B) Myc-tagged HA95 was immunoprecipitated from Bjab cells stably expressing HA95–Myc using anti-Myc antibodies, and precipitates were immunoblotted using indicated antibodies. Control immunoprecipitations were done with preimmune rabbit IgGs. (C) HA95-IPs were extracted with 0, 0.25, 0.5, 0.75, 1.0, or 1.25 M NaCl before sedimentation and immunoblotting of pellets and supernatants.
LAP2β interacts with HA95 via two distinct domains. (A) GST–LAP2β deletion peptides. (B) Indicated peptides were incubated in nuclear extracts of Bjab cells expressing HA95–Myc and sedimented by GST precipitation. Binding of HA95–Myc to peptides was analyzed by immunoblotting using anti-Myc antibodies. Control incubations contained GST or glutathione beads alone. (C) GST–LAP2β(137–373) was incubated in a nuclear extract of Myc-tagged p70S6 kinase-expressing cells, and binding (or lack thereof) of p70S6 kinase to the peptide was detected by GST precipitation and immunoblotting. (D) GST–LAP2β overlays of immunoprecipitated HA95 or B-type lamins. Peptide binding was detected using anti-GST antibodies. (E) Peptide-mediated dissociation of LAP2β from HA95. HA95-IPs were incubated with 100 μM GST–LAP2β peptides, HA95-IPs were sedimented, and pellets and supernatants were immunoblotted using the indicated antibodies. Arrows indicate incubation with LAP2β(137–298) followed by addition of LAP2β(299–373) (lanes 7 and 8) or vice versa (lanes 9 and 10).
Disruption of HA95 interaction with LAP2β does not interfere with nuclear assembly in vitro. (A) Purified HeLa nuclei (Nuc) were disassembled in mitotic extract, and chromosomes (Chr) were immunoblotted using the indicated antibodies. (B) Indicated GST–LAP2β peptides were allowed to bind to chromosomes, and binding was analyzed by immunofluorescence using anti-GST antibodies. Insets, DNA labeled with Hoescht 33342. (C) Chromosomes harboring the indicated peptides were incubated in interphase extract under conditions promoting nuclear formation. Membrane assembly was examined by DiOC6 labeling and phase contrast microscopy. (D) At the end of incubation, the nuclear distribution of GST–LAP2β peptides, LAP2β, A- and B-type lamins, and BAF was examined by immunofluorescence. (E) Nuclei or chromatin masses were also sedimented through sucrose, and proteins were immunoblotted using the indicated antibodies. Bars, 10 μm.
Characterization of nuclei used in the in vitro DNA replication assay. (A) Nuclei purified from HeLa cells in G1 phase were loaded with GST–LAP2β(1–452). Peptide uptake was analyzed by immunofluorescence using anti-GST antibodies. (B) GST–LAP2β(1–452)-loaded G1 nuclei were exposed to an S-phase extract, and import of Cdc6 was visualized by immunofluorescence. Note that the G1 nuclei contain low detectable levels of Cdc6. (C) Cdc6 import was also examined in nuclei containing the indicated peptides. G1 nuclei were also analyzed by (D) TUNEL and (E) electrophoresis to monitor DNA degradation after exposure to replication extract at 4°C or 37°C. Bars, 10 μm.
LAP2β(137–298) inhibits initiation of DNA replication in G1 nuclei. (A) Peptide-loaded G1 nuclei were incubated in S-phase extract containing [α32P]dCTP, dNTPs, GTP, and an ATP-regenerating system. [α32P]dCTP incorporation was analyzed by phosphorImager (cpm/105 nuclei). (B) Nuclei isolated from HeLa cells at the indicated cell cycle stages were incubated in extracts from S or G0 cells under conditions promoting replication. S-phase extracts also contained 1 mM olomoucine (+Olo) or H2O (−Olo) (right panel). Synthesized DNA was analyzed by autoradiography. (C) Relative BAF content of G1 nuclei loaded with the indicated peptides and exposed to S-phase extract was examined by immunoblotting. HA95 was also detected as a loading control in the gel. (D) Summary of GST–LAP2β peptides harboring HA95-NBD supporting (+) or inhibiting (−) replication in G1 nuclei. (E) BrdU density substitution experiment. G1 nuclei containing either no peptide (circles), GST alone (triangles), or GST–LAP2β(137–298) (squares) were allowed to replicate in S-phase extract containing BrdU and [α32P]dCTP. Substituted DNA was separated by centrifugation in CsCl gradients. cpm of each fraction was plotted against fractions of equal densities. HL indicates density of heavy-light replicated DNA. (F) Nuclei from S-phase HeLa cells were loaded with GST–LAP2β peptides and peptide uptake was analyzed as in Fig. 4 A (GST–LAP2β[1–452] is shown). Peptide-loaded nuclei were incubated in S-phase extract and incorporation of [α32P]dCTP was measured by phosphorImager (cpm/105 nuclei). Bar, 10 μM.
Dissociation of HA95 from HA95-NBD in G1 elicits degradation of Cdc6. (A) HA95 (top three panels) or Cdc6 (bottom three panels) was immunoprecipitated from G1- or S-phase HeLa cells, and immune precipitates (P) and supernatants (S) were immunoblotted using either precipitating antibody. (B) Nuclei from G1-phase cells (lane 1) or G1 nuclei loaded with the indicated GST–LAP2β peptides (lanes 2–5) and incubated in nuclear isolation buffer for 1 h were immunoblotted using the indicated antibodies. In lanes 6–8, G1 nuclei loaded with LAP2β(137–298) were incubated in buffer for 1 h together with 25 μM β-lactone, LLnL, or LLM. In lane 9, whole samples (WS; nuclei + buffer) were immunoblotted. (C) G1 nuclei were loaded with GST–LAP2β(137–298) or GST–LAP2β(299–373) in the presence of no inhibitor (−), β-lactone, LLnL, or LLM. Nuclei were allowed to replicate in S-phase extract containing [α32P]dCTP, and synthesized DNA was analyzed by autoradiography.
Nuclear injection of GST–LAP2β(137–298) in G1 does not alter distribution of NE proteins or BAF. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone, cultured for 2–3 h, and analyzed by immunofluorescence using anti-GST, LAP2β, lamin B (goat polyclonal), or BAF antibodies. Arrows point to noninjected cells. Bar, 10 μm.
Nuclear injection of GST–LAP2β(137–298) in G1 inhibits replication. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone together with a 150-kD FITC–dextran to visualize injections, and cells were cultured for 9–10 h with BrdU. Mock (buffer)-injected cells were also cultured with BrdU and 50 μM aphidicolin. BrdU incorporation was detected using TRITC-conjugated anti-BrdU antibodies. DNA was labeled with Hoechst 33342. (B) Percentages of cells labeled with BrdU 9–10 h after peptide injection (n = 45–50/treatment in two replicates). (C) Immunofluorescence analysis of Cdc6 and Orc2p in G1 cells injected as in A. Cells were fixed 2 h after peptide injection. Arrows point to noninjected cells. Bars, 10 μm.
Functional domains of LAP2β. Identified functional regions of LAP2β (LEM and transmembrane [TM]) and domains binding to indicated ligands are shown. Numbers refer to amino acid positions. See text for references to binding domains.


Contemporary systems are extremely effective simply because they depend on among the most effective genetic tendencies we do have – the choice for data that is visually displayed. The mind includes a great prejudice for data that is visually displayed. Films, tv, films, & most computer applications are extremely visually-oriented and so attract and keep up with the interest of young kids. They neglect to create additional ideas while young kids sit-in top of tv all night. However the systems that reward young kids the best are the ones that are fun and permit the kid to build up thinking skills and their attention, problemsolving.
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A tendon is the anatomical term used for the connective tissue which binds muscles to their attachments. Normally, muscles are attached directly to bone at either end. However, in some cases, tendon attachments are made with ligaments, joint capsules and strong layers of soft tissue known as fascia whose role it is to provide compartments within the body and strength.
Tendonitis is a familiar term used to describe pain and inflammation to any tendon. Tendinitis is also referred to as tendinopathy. As different muscles within the body have varied roles so do their respective tendons. Therefore, one singler cause of tendonitis does not exist and is often related to the particular body area affected.
Common types of tendonitis and their causes?
The Achilles Tendon attaches the calf muscles to the heel bone and has a spring like structure which provides efficiency, particularly during walking and running. Unaccustomed increases in these activities particularly for individuals who walk/run with excessive rolling in of the feet (over-pronation) may cause this condition.
Patellar Tendonitis is also known as "jumper's knee" as this tendon bears several times the bodyweight when landing. As this pseudonym suggests this condition is almost exclusively seen in sportsmen/sportswomen involved in athletics, basketball, netball and sometimes football.
The tendons of the rotator cuff muscles which function to move the shoulder joints may suffer with pain and inflammation as a secondary consequence of age related processes to the shoulder/s or due to being compressed repeatedly as the arm is lifted.
These conditions are named after their respective tasks due to the significant load each tendon undergoes performing these tasks. However, the most common cause of these conditions remains repetitive computer work with an incorrect ergonomic setup.
Tendonitis results in pain and swelling which can normally be felt to the affected tendon. Normally the middle portion of the tendon is affected but in some cases the symptoms can occur right at the insertion point of the tendon (insertional tendonitis).
Pain is felt when the affected muscle and tendon is being placed under load. In most cases, the symptoms ease off after a few minutes of an aggravating activity and then return if the activity is prolonged.
In rare cases of severe pain from tendonitis, the affected limb may begin to feel weak with certain tasks.
Physiotherapy is normally the first line of treatment for tendonitis. This normally involves appropriate stretches and placing a gradual amount of weight through the affected tendon with strengthening exercises.
For patients who have not found physiotherapy has enabled a return to normal function or sports, Circle Health is able to offer appointments with expert consultant orthopaedic surgeons or sports medicine consultants at short notice. Under these circumstances, our consultants are able to best guide management following an expertly conducted physical examination and imaging, normally in the form of an ultrasound or MRI scan.
Historically, patients with persistent tendonitis may have been offered corticosteroid injections. This is is now performed in very rare circumstances as evidence indicates there may be some negative side effects for tendons in the longer term. PRP injections may sometimes be offered as a treatment instead. Blood plasma (which is taken from patients through a small blood test, filtered and stored in a lab before the injection) contains natural substances which encourages healing without any obvious negative effects.
Shockwave therapy is a form of therapeutic ultrasound which delivers large amounts of mechanical "shockwaves" to the affected tendon. The intention is to induce a low level trauma to the tendon (which has failed to heal) in order to kickstart a healing response. As it is an invasive form of treatment, doses are spaced over a number of weeks. This gives the affected tendon time to strengthen itself between doses to limit the possibility of any damage (tear or rupture) to the tendon.
Although surgery is a relatively safe procedure with very small incisions being needed, there is still a small risk of infection. Due to this and with the development of the above treatments, surgery for tendinitis is offered only as a last resort.
Surgery involves identifying the exact location of pain and swelling to the tendon with a physical examination and diagnostic imaging. The swelling to this area is formed by an increase in micro blood vessels and calcific deposits. These pathological parts of the tendon are then resected leaving the healthy piece of tendon intact.


Building a reputation as an eco-conscious company carries a great deal of weight with today's green-minded consumers. Carolina Fibre offers paper recycling, aluminum recycling, consulting services, confidential shredding and plastic recycling to help businesses become more sustainable. Plastic recycling is a budget-friendly, responsible way to dispose of post-industrial waste. Plastics are known as the largest contributor of waste in the world and take hundreds of years to biodegrade.
Polythylene terephthalate (PET, PETE) – PET is used in synthetic fibers like beverage, food and other liquid containers.
High-density polyethylene (HDPE) – HDPE is made from petroleum and commonly used in the production of plastic bottles. Some of its many common applications include folding chairs and tables, hard hats, plastic bags, plastic lumber and wood plastic composites.
Polyvinyl chloride (PVC) – Just about half of the world's polyvinyl chloride resin manufactured annually is used for producing pipes for municipal and industrial applications.
Low-density polyethylene (LDPE) – LDPE is widely used for parts that need to be weldable and machinable, and for manufacturing various containers, tubing, plastic bags for computer components, and molded laboratory equipment.
Polypropylene (PP) – You'll find PP in packaging and labeling, and things like textiles, carpets, stationary, and lab equipment.
Polystyrene (PS) – PS is unfortunately very slow to biodegrade and is often abundant as a form of littler in the outdoor environment. It's the most widely us plastics and includes protective packaging, containers, lids, bottles, trays and disposable utensils. Discarded PS does not biodegrade for hundreds of years.
Other (often polycarbonate or ABS) – Polycarbonate is used for electronic applications, data storage, construction materials and eye protection. Acrylonitrile butadiene styrene (ABS) is used for medical devices for blood access, enclosures for electrical and electronic assemblies, protective headgear. ABS can be recycled, but it is not accepted by all recycling facilities.
Plastics are not recycled as efficiently as they should be. And with the use of plastics increasing over the year—in everyday products, furniture, and appliances—it's more important than ever for your business to conserve energy and reduce your own destructive waste output. Aside from the positive impact you'll have on the environment, going green is good for your business. Carolina Fibre can help your organization with its sustainability efforts to lower operating costs, attract new business and reduce waste.


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Carla Williams (nee Green) is the athletic director of the Virginia Cavaliers. She is the first African American woman in the Power Five conferences to hold this position.

Early life
Williams was born to parents Barner and Johnnie Mae in LaGrange, Georgia. In middle school, she played quarterback and running back in organized football, as well as basketball and softball. While attending LaGrange High School, Williams played point guard for the Georgia Metros. Her coaches would drive her two hours there and back to practice. During her high school career, Williams led LaGrange to two Class AAAA state titles and was named the most valuable player of the 1985 Georgia All-Star game.
During her senior year of high school, Williams was approached by Andy Landers to recruit her for the women's basketball program at the University of Georgia (UGA).

Collegiate career
After graduating from LaGrange, Williams went to UGA on an athletic scholarship and made the starting lineup in her sophomore year. During that season, she was named to the South squad for the United States Olympic Festival in Houston. She later asked Landers why he offered her a scholarship over other players that were ranked higher and he told her "you have all the intangibles that we need for our program." In 1987, Williams was again named to the United States Olympic Festival and Atlanta Tipoff Club's Division I women's basketball All-Star Team. Upon completing her Bachelor of Arts degree in sociology in 1989, Williams was a four-year letterwinner and finished her career with 1,115 points. She was also an All-SEC selection guard and tenth highest scorer in UGA women's basketball history.

Career
After graduating from UGA in 1989, Williams competed in professional basketball in Spain, but was encouraged by Jerry Legge to return to her alma mater to complete a Master's degree of Public Administration. After completing her MPA, Williams became an assistant coach for the Lady Bulldogs until 1996 under Andy Landers, who she played for as an athlete. During her time as a coach, she helped the Lady Bulldogs advance to consecutive Final Fours in 1995 and 1996, and the '96 national championship game against Tennessee. After five years in this role, she moved to Florida State University (FSU) when her husband accepted a job there, taking a pay cut to work an entry-level position in the student services office.

During her time at FSU, Williams also completed her doctorate in sports administration and volunteered for the Florida State Seminoles Director of Athletics Dave Hart in several areas in the department including radio and television broadcasts. Upon finishing her degree, Williams accepted a position at Vanderbilt University as an associate athletics director and senior women's administrator where she oversaw varsity women's basketball, men's and women's tennis, women's soccer, the Hendrix Dining Room and video operations. In 2004, Williams returned to Georgia as an associate director of athletics before becoming the deputy athletics director.

As the athletics director of the University of Georgia, Williams served on the executive committee of both the National Association of Collegiate Directors of Athletics and the National Association of Collegiate Women Athletic Administrators, as well the NCAA's Woman of the Year Selection and Women's Basketball Issues Committees. In recognition of her efforts, she was named one of the top-10 Senior Woman Administrators in the NCAA. She was also the recipient of the Athens Woman of Distinction by the Girl Scouts of Historic Georgia.

After spending 13 years in athletic administration at Georgia, Williams became the first African-American woman to lead an athletic department at a Power 5 school (ACC, SEC, Big Ten, Big 12 and Pac-12). She joined the faculty at the University of Virginia as an athletic director in 2017. She began her tenure at the institution by announcing The Master Plan, a project to "address the immediate needs to upgrade facilities for the Cavalier football program and UVA Olympic Sports." In recognition of her efforts, Williams received UGA's Bill Hartman Award as someone who has "demonstrated excellence in their profession and/or in service to others by 20 or more years of superior performance after graduation."

Personal life
Williams and her husband, Brian, have three children together.

References

External links
 Virginia profile

Year of birth missing (living people)
Living people
American women's basketball players
Georgia Lady Bulldogs basketball players
Vanderbilt University staff
Virginia Cavaliers athletic directors
People from LaGrange, Georgia
Basketball players from Georgia (U.S. state)
African-American college athletic directors in the United States
Women college athletic directors in the United States

redpajama_set_name: RedPajamaWikipedia


text:
Record Office lays on art activities for children and young people
30 March 2021 By Joanne Thompson
Youngsters can enjoy fun art activities in the comfort of their own home this Easter and earn a qualification at the same time.
Durham County Council's Durham County Record Office is using funding from Arts Council England to give children and young people the chance to get creative online. Next week families can sign up for two free online craft workshops for inspiration towards gaining their 'Arts Award Discover.'
Arts Award is a series of qualifications administered by Trinity College London for children and young people aged five to 25. Discover is the first level.
Children and young people can gain the qualification by downloading a logbook from the record office website at Arts Award. The website also includes a series of short videos filmed by record office staff, guiding families through the award.
Children and young people can then work through the suggested creative activities, including designing an illuminated letter and creating an illustrated diary entry, both inspired by real examples from the archive collections.
Youngsters who fill in the logbook should return it to the record office. It will then be submitted to Trinity College London (TCL) and children will receive an official certificate.
The Arts Council England funding will allow the first 50 children and young people to return their logbooks and have them moderated free of charge.
After this, a fee will be charged, to cover TCL's normal moderation costs.
Families can sign up for two online craft workshops, aimed at primary school aged children, over the Easter holidays.
'Snippets from the archives: Investigating Easter in the past' will look at examples from the record office's archive collections to show how Easter was celebrated in the past. There will also be a craft activity.
The workshops will last about 30 minutes and run on Tuesday 6 April at 10am and Thursday 8 April at 2pm.
The sessions are free but places must be booked in advance, online at Events
Education archivist Dawn Layland said: "The activities in the Arts Award logbook are a great way for children and young people to get creative while enjoying themselves over Easter, and all can be done from the comfort of their own home.
"The same goes for the workshops which offer primary school aged children the chance to find out what Easter was like in the past.
"We're grateful to Arts Council England for the funding we've been given and pleased to be able to use it to help young people gain a useful qualification."
Further information about Arts Awards can be found at www.artsaward.org.uk.

redpajama_set_name: RedPajamaCommonCrawl


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Google's Cardboard device for virtual reality applications, unveiled at Google I/O in 2014.
Developers creating content for Google's Cardboard virtual-reality system should look to short online videos for inspiration and avoid drawn-out experiences.
Content for Cardboard should be "snackable virtual reality," said Jon Wiley, the product's principal designer, during the I/O developer conference on Friday.
"For Cardboard, you want [an experience] to be more like traditional YouTube content," he said, adding that Google's VR platform isn't really designed for "long duration experiences."
Google sees Cardboard as a device that friends can pass around to get a feel for what VR is all about, said Manuel Clement , a user experience designer on Google's virtual reality team. In that context, shorter content would work better.
While Google's rivals in VR and augmented reality have built polished, sleek headsets, don't expect Cardboard to get a high-end makeover any time soon.
"Part of the magic of Cardboard is the simplicity of the system," said Wiley, adding that Google may look to simplify the platform even more.
Although Cardboard is just a simple box with a plastic lens and a smartphone pushed inside, it can still provide a great experience if developers don't push beyond its limits, Wiley said. Developing apps that are too complex or sophisticated will exceed its capabilities.
To incorporate virtual reality into their apps, Google recommends developers start small and consider how introducing a 3D component will make their program better.
Allowing people to tour a house in 3D instead of just viewing a photo gallery would give a real estate app an advantage, said Erica Morse, a Cardboard user experience designer.
Developers must also remember that VR content can make people feel disoriented, so they need to take into account the physiological response to what people see, said Alex Faaborg, who works on the virtual reality design team. That means creating apps that allow the display and the person's body to work together.
Apps should maintain a stable horizon line, and head tracking needs to remain engaged so people can navigate environments smoothly. Uneven acceleration can lead to nausea, and if developers can't create seamless transitions, Faaborg suggests fading to black, or using audio to help with the transition to a new scene.
Some users might just hold Cardboard in front of their faces because they don't know they can move their head around to explore a virtual setting. To get people more engaged, developers could place an object off to the side so that users are encouraged to turn their gaze, he said.
And developers should think visually about displaying information, Faaborg said. If an app features a spaceship that's sustained heavy damage, don't use a bar graph to convey the information; show the damage on the ship's wing.
To help developers build VR apps, Google created Cardboard Design Studio, an app that explains both simple and advanced techniques.


LONDON, Nov 6 (Reuters) – Oil prices fell on Tuesday after Washington granted sanctions exemptions to top buyers of Iranian oil, lifting supply concerns and turning the market's focus to worries that an economic slowdown may curb fuel demand. U.S. West Texas Intermediate crude futures were at $62.80 a barrel, down 30 cents from their last settlement.
Benchmark Brent crude futures were down 53 cents at $72.64 a barrel by 1243 GMT.
Washington gave 180-day exemptions to eight importers – China, India, South Korea, Japan, Italy, Greece, Taiwan and Turkey. This group takes as much as three-quarters of Iran's seaborne oil exports, trade data shows, meaning Iran will still be allowed to export some oil for now.
Iran's crude exports could fall to little more than 1 million barrels per day (bpd) in November, compared with a 2018 high of around 2.6 million. But that figure could rise from December as importers use their waivers.
China was given a waiver to import around 360,000 bpd from Iran during the exemption period, sources told Reuters.
"The Iran factor continues to occupy the minds of market participants. That said, it is failing to spur buying pressures," PVM said in a note.
Meanwhile, concerns about demand continue. The trade dispute between the United States and China threatens growth in the world's two biggest economies and currency weakness is pressuring economies in Asia, including India and Indonesia.
On the supply side, oil is ample despite the sanctions against Iran as output from the world's top three producers – Russia, the United States and Saudi Arabia – is rising.
The three countries combined produced more than 33 million bpd for the first time in October, meaning they alone meet more than a third of the world's almost 100 million bpd of crude oil consumption.
Hedge fund managers were net sellers of petroleum-linked futures and options last week, taking their net long position to the lowest level in 15 months, according to records published by regulators and exchanges.


text:
Spirit of The South
This years headliners BLACKBERRY SMOKE
This years main support The Allman Betts Band
with guests The Wild Feathers
The Allman Brothers Band Museum and Tribute
We Believe Saint John
This years headliners
Chris Lane's star continues to rise with 1.1 BILLION on-demand streams, two #1 hits – PLATINUM "Fix" + 2X PLATINUM "I Don't Know About You" – and two Top 10 albums to his credit. Stacking his milestones with a pair of all-star collaborations, Lane teamed up with LA-based artist/producer Gryffin on the infectious track "Hold You Tonight" after his smash PLATINUM single "Take Back Home Girl" with Tori Kelly. Lane has been spotlighted in PEOPLE's 2019 Sexiest Man Alive issue, scored nominations at the iHeartRadio Music Awards, ACM Awards and Radio Disney Music Awards, plus appeared on TODAY, The Bachelor, The Tonight Show Starring Jimmy Fallon, Late Night with Seth Meyers and CONAN. After sharing the bill with Florida Georgia Line, Brad Paisley, Dan + Shay and more A-list acts, the Kernersville, NC-native's headline BIG, BIG PLANS TOUR touted 25 sellouts in major cities coast-to-coast. Lane married Lauren Bushnell in 2019, after popping the question with "Big, Big Plans" – his Top 5-and-rising, GOLD-certified Country radio single.
Singer-songwriter MacKenzie Porter is a rising Country talent whose sound knows no borders. Signed to Big Loud Records, Porter is surging now with DRINKIN' SONGS: THE COLLECTION, an arsenal of two years' worth of new music produced by Joey Moi (Jake Owen, Morgan Wallen). Since moving to Nashville in 2014 from her native Canada, she's developed a radiant Country style which threads the needle between traditional heart and modern energy, often collaborating with songwriting mainstays like Nicolle Galyon, Craig Wiseman, Natalie Hemby, and Tommy English. Her "mesmerizing vocals and hook-driven songs" (Billboard) have sparked a list of headline-worthy accolades, including multiple Canadian Country Music Award nominations — 4X Female Artist of the Year, Songwriter of the Year and Fans' Choice — as well as a previous JUNO Award nomination for Country Album of the Year. Making her mark, Porter became the first female artist to have three back-to-back #1 singles at Canadian Country radio in 22 years with "About You," "These Days" and "Seeing Other People" in early 2020, following Shania Twain in 1998. Porter also earned her first crossover Top 10 at Canadian pop-radio — a first since 2003 for a Canadian female country artist — with "These Days (Remix)." She's toured extensively on her own, sharing the stage with Kenny Chesney, Rascal Flatts, Chris Lane, Blake Shelton and Dallas Smith, and co-starred in the Netflix series, "Travelers."
Love, Heartbreak, & Everything in Between
Born and raised in the small prairie town of Weyburn, Saskatchewan, Tenille Arts picked up the guitar and penned her first song at age 13 and has been performing nonstop ever since. She made her first appearance on The Bachelor in January 2018, which resulted in a record deal with powerhouse indie label Reviver Records. She made her second appearance on the show in January of 2019 performing "I Hate This." The song hit #1 on the U.S. and Canadian iTunes Country Charts and #9 U.S. iTunes all-genre. Her third appearance on The Bachelor preceded the release of her new album Love, Heartbreak, & Everything In Between in January of 2020.
Tenille's music has tallied over 51 million lifetime streams. Pandora and CMT listed her as one of their new artists to watch, and Rolling Stone called Tenille "An edgy, Fearless-era Taylor Swift with crystalline vocals; ideal for hopeless romantics who are fire signs."
Tenille made her Grand Ole Opry debut on September 21, 2019 and has made appearances on the TODAY Show, the Radio Disney Music Awards and sang the Canadian National Anthem at game 3 of the 2019 NBA finals and at the 2020 NBA All-Star Game.
Neely G Entertainment 2019


text:
Internet Explorer flaw puts XP users especially at risk.
If you haven't upgraded to a modern operating system just yet, now is likely the time. Excerpt from an Engadget article:
In a security alert released on Saturday, Microsoft reports that there's a serious vulnerability in Internet Explorer 6 through 11 that could allow hackers to take over your computer remotely if you happen to visit a malicious website. According to security firm FireEye, it has already found evidence of an attack that targets IE 9 through 11 that uses a well-known Flash exploitation technique to gain access to your computer's memory. Microsoft has already said it plans to roll out an IE security update for all modern versions of Windows, but if you're using XP, well, you're out of luck, as support for that 12-year-old OS ended a few weeks ago.
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text:
Turkish court frees key corruption suspects
Court releases five people – including two ministers' sons – embroiled in graft probe that has rocked government.
Baris Guler (in sunglasses) was among those released after the necessary evidence had been collected [Reuters]
Published On 28 Feb 201428 Feb 2014
A Turkish court has released five people, including the sons of two ministers, who were detained on corruption allegations in mid-December in a high-profile probe that has rocked the government, according to local media.
The private NTV channel reported on Friday that Baris Guler, the son of the former interior minister, as well as Kaan Caglayan, the son of the ex-economy minister, and Azerbaijani businessman Reza Zarrab, were among those freed.
NTV said the suspects were released because the "necessary evidence had been collected".
Guler and Caglayan had been charged with acting as intermediaries for giving and taking bribes, while Zarrab was suspected of forming a ring that bribed officials to disguise illegal gold sales to sanctions-hit Iran via state-owned Halkbank.
The men walked free two weeks after Suleyman Arslan, the former chief executive of Halkbank, who was also caught up in the police raids, was released.
Arslan was accused of corruption, fraud and money laundering in connection with illegal gold sales to Iran.
The graft scandal has struck at the heart of Prime Minister Recep Tayyip Erdogan's government, posing the biggest challenge yet to his 11 years in power.
The crisis has prompted a cabinet reshuffle and the two ministers whose sons were implicated in the scandal have resigned.
Erdogan has accused supporters of exiled Muslim leader Fethullah Gulen, who wields considerable influence in the judiciary and police, of launching the probe as part of a plot against his government ahead of local polls in March.
Erdogan has retaliated by sacking or reassigning hundreds of police and prosecutors believed to have links to Gulen.


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text:
The growth in newspaper coverage of tobacco control in China, 2000-2010
BMC Public Health. 2012 Mar 7;12:160. doi: 10.1186/1471-2458-12-160.
Junling Gao 1 , Simon Chapman, Shaojing Sun, Hua Fu, Pinpin Zheng
1 School of Public Health, Fudan University, Key Laboratory of Public Health Safety, Ministry of Education, Shanghai 200032, China.
DOI: 10.1186/1471-2458-12-160
Background: Media coverage of tobacco-related issues can potentially shape individual beliefs, attitudes and behaviors about tobacco use. This study aims to describe news coverage of tobacco control related issues in Chinese newspapers from 2000 to 2010.
Methods: All 1149 articles related to tobacco control were extracted from the Database of Chinese Important Newspapers and content analyzed for the period Jan 1, 2000 to Dec 31, 2010. The changing pattern of tobacco control topic, article type, viewpoint, and article origin, and their relationship were analysed.
Results: News coverage of tobacco control related issues increased significantly (p < 0.01) from 2000 to 2010, with news coverage being relatively intensive in May and June (p < 0.01), around World No Tobacco Day. 24.9% (n = 286) of all articles focused on secondhand smoke, 25.3% (n = 291) warned about the dangers of active smoking, and 10.0% (n = 115) focused on prevention and cessation programs and campaigns. Tobacco control topics varied significantly between national vs city/regional newspapers (χ2 = 24.09, p = 0.002) and article types (χ2 = 193.35, p < 0.001). Articles in national newspapers had more coverage of the dangers of tobacco and on enforcing bans on tobacco-advertising. News stories centered around monitoring tobacco use and smoke free activity, while editorials focused on enforcing bans on tobacco-advertising, youth access and programs and campaigns. Letters to editors focused on the dangers of smoking, raising tax, and smoking cessation. More articles (50.4%) took an anti-tobacco position (compared with 10.5% which were pro-smoking), with the amount of negative coverage growing significantly across the decade. National articles tended to lean toward anti-tobacco, however, local articles tended mix of pro-tobacco and neutral/balance positions. Editorials seemed to be more anti-tobacco oriented, but letters to the editor tended to show a mix of anti-tobacco and pro-tobacco positions.
Conclusion: Chinese newspapers are giving increasing attention to tobacco control, but coverage remains lower than in the USA and Australia. Health workers need to give higher priority to efforts to increase news coverage beyond the present concentration around World No Tobacco Day and to develop strategies for making tobacco control issues more newsworthy to both national and local news outlets.
Newspapers as Topic / statistics & numerical data*
Smoking Prevention*


Glaziers Westcombe Park is well known in the area for it's highly responsive and cost-effective services for urgent glass repair, replacement and installation.
Glaziers Westcombe Park is well known in the area for its highly responsive and cost-effective services for urgent glass repair, replacement and installation.
However, we also have a strong reputation for providing versatile and professional services for home improvement, building and maintenance projects in Westcombe Park, SE3 too. This includes offering highly specialist supply and installation of glazing products such as crushed and cracked ice glass.
Where can you get crushed and cracked ice glass in Westcombe Park, SE3??
Thats because Glaziers Westcombe Park has extensive experience, high levels of training and the latest technology we can draw on.
To source crushed and cracked ice glass and arrange installation in any address in Westcombe Park, SE3, simply use our handy instant quote system or give us a call. Our site visits are free of charge and we would be pleased to provide a fixed price quote you can rely on.
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Glass Westcombe Park, SE3 pipes must be cleaned periodically. They are also great for germaphobes. They produce cleaner smoke than any other material, and they allow the smoker to see the smoke as it is pulled into and out of the pipe. There are two fundamental procedures to clean glass Westcombe Park, SE3 pipes.
Thankfully the notion of making cocktail ice is really straightforward, but understanding what you're attempting to achieve when cracking ice is a bit more complex. For those who have space get a more compact tub for crushed ice otherwise maintain a ziploc bag of crushed in the principal tub. To reduce mild pain, you may apply on the affected place. The lower part of a bar where extra alcohol is stored is referred to as the doghouse. Rather than dipping, roll the border of the glass Westcombe Park, SE3 so the edging sticks only to the outside. Yes you may use the rear of a bar spoon or a different product, but should you wish to simplify the procedure and look cool doing it I recommend the Tap Icer.


text:
Die Egypt International 2019 im Badminton fanden vom 17. bis zum 20. Oktober 2019 in Kairo statt.

Sieger und Platzierte

Weblinks
 tournamentsoftware.com

2019
Badminton 2019
Badmintonwettbewerb in Kairo


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Sinisa Krajnovic, head of development unit networks at Ericsson.
Ericsson announced Thursday plans to open a design center in Austin with 80 employees by next June.
The center is focused on microelectronics and creating products for the 5G telecommunications marketplace.
"This R&D center will be one of our key centers when it comes to 5G ASIC development," said Sinisa Krajnovic, head of development unit networks at Ericsson.
It's Ericsson's third global design center. The telecommunications giant, which has its North American headquarters based in Plano, Texas, also has a design center at its global headquarters in Stockholm, Sweden and in China.
This center will work closely with those other teams, but after a worldwide search, Ericsson selected Austin because of the telecommunications and semiconductor talent here and the city's technology ecosystem, Krajnovic said.
The Austin team will be developing the core microelectronics for Ericsson's 5G next-generation products.
"They are truly the brain for our mobile infrastructure," Krajnovic said.
The Austin ASIC Design Center is open now and recruitments are ongoing, Krajnovic said.
ASIC stands for Application Specific Integrated Circuit. Such microelectronics are at the core of all Ericsson Radio Systems and can be seen as processors that are specially made for the computation needs of mobile infrastructure. Ericsson's processors are 100 times faster and more cost-efficient than general off the shelf processors, Krajnovic said. It's a key competitive advantage to have application specific processors, he said.
Ericsson is hiring experienced ASIC developers and architects, Krajnovic said.
Austin has the top experts in this ASIC domain. Austin is also home to a lot of Ericsson's suppliers and partners.
Ericsson has about 15 employees in Austin currently working on customer-specific projects, but this will be the company's first major site in Austin.
Krajnovic said Ericsson is confident they will find the workers they need in Austin.
Ericsson plans its first commercial 5G product coming out in 2019 and for deployment that year, Krajnovic said.
"Those teams in Austin will be a very important part of that journey," he said.
Ericsson has temporary offices for the design center while it's looking for a permanent site near Austin's other processor manufacturers, Krajnovic said. It will be working closing with the major Silicon fab houses in Austin, he said.
For more information on the jobs, go to the Ericsson career site and search for Austin.