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Copyright © 2012 The Authors All rights reserved.
Cell Reports, Volume 2, Issue 5, 1316-1328, 01 November 2012
Presenilin Deficiency or Lysosomal Inhibition Enhances Wnt Signaling through Relocalization of GSK3 to the Late-Endosomal Compartment
Radek Dobrowolski1, 3, 4, Philipp Vick1, 3, Diego Ploper1, Iwona Gumper2, Harriet Snitkin2, David D. Sabatini2 and Edward M. De Robertis1, ,
1 Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USA
2 Department of Cell Biology, New York University School of Medicine, New York, NY 10016-6497, USA
3 These authors contributed equally to this work
4 Present address: Department of Biological Sciences, Rutgers University, Newark, NJ 07102, USA
Graphical Abstract
► Presenilin (PS) depletion leads to changes in the endolysosomal pathway ► Multivesicular endosomes are increased in PS-deficient cells ► Wnt signaling is enhanced in PS-depleted cells via increased GSK3 sequestration ► Higher Wnt signaling leads to increased stability of GSK3 protein substrates such as Tau
- Sustained canonical Wnt signaling requires the inhibition of glycogen synthase kinase 3 (GSK3) activity by sequestration of GSK3 inside multivesicular endosomes (MVEs). Here, we show that Wnt signaling is increased by the lysosomal inhibitor chloroquine, which causes accumulation of MVEs. A similar MVE expansion and increased Wnt responsiveness was found in cells deficient in presenilin, a protein associated with Alzheimer's disease. The Wnt-enhancing effects were entirely dependent on the functional endosomal sorting complex required for transport (ESCRT), which is needed for the formation of intraluminal vesicles in MVEs. We suggest that accumulation of late endosomal structures leads to enhanced canonical Wnt signaling through increased Wnt-receptor/GSK3 sequestration. The decrease in GSK3 cytosolic activity stabilized cytoplasmic GSK3 substrates such as β-catenin, the microtubule-associated protein Tau, and other proteins. These results underscore the importance of the endosomal pathway in canonical Wnt signaling and reveal a mechanism for regulation of Wnt signaling by presenilin deficiency.
Canonical Wnt signaling is essential for embryonic development, stem cell and tissue homeostasis, and regeneration in adults (MacDonald et al., 2009; Angers and Moon, 2009). Aberrant Wnt signaling has been associated with human diseases such as cancer, bone disorders, and neurodegeneration (Clevers and Nusse, 2012; Boonen et al., 2009). In the absence of Wnt ligands, the adaptor protein and transcription cofactor β-catenin is phosphorylated by GSK3 in a destruction complex consisting of the tumor suppressor adenomatous polyposis coli (APC), Axin, casein kinase 1 (CK1), and the E3-polyubiquitin ligase βTrCP (Cadigan and Peifer, 2009; Li et al., 2012). Phosphorylations by GSK3 target β-catenin and other proteins for polyubiquitinylation and degradation in the proteasome (Kim et al., 2009; Taelman et al., 2010; Clevers and Nusse, 2012). Binding of Wnt ligands to their receptors Frizzled (Fz) and LDL-receptor related protein 5/6 (LRP5/6) triggers recruitment of Dishevelled (Dvl), Axin, and GSK3 to the plasma membrane (Bilic et al., 2007; Zeng et al., 2008). GSK3 is first recruited by the binding of Axin to LRP6 and then becomes engaged in the phosphorylation of LRP6, Fz, Dvl, Axin, and β-catenin, which contain multiple GSK3 sites, explaining the requirement of an intact GSK3 catalytic site for its relocalization (Taelman et al., 2010). Wnt receptor complexes containing Axin and GSK3 are then internalized into the cell by endocytosis (Blitzer and Nusse, 2006; Yamamoto et al., 2006) and subsequently sequestered by incorporation into the intraluminal vesicles (ILVs) of late endosomes that are produced by invagination and scission from the endosomal limiting membrane (Taelman et al., 2010; Dobrowolski and De Robertis, 2012). Sequestration of active GSK3 inside multivesicular endosomes (MVEs) leads to sustained stabilization of the half-life of many GSK3 protein substrates (Taelman et al., 2010), principal among which is newly synthesized β-catenin, which enters the nucleus to coactivate Wnt target genes.
The integration of cell signaling and endocytosis is critical for signal transduction outcomes (Sorkin and von Zastrow, 2009; Dobrowolski and De Robertis, 2012). Although most receptor complexes are negatively regulated by endocytosis (Katzmann et al., 2002), Wnt signal transduction requires endocytosis (Blitzer and Nusse, 2006). Inhibition of ILV formation in MVEs (also referred to in the literature as multivesicular bodies [MVBs]) by interfering with components of the endosomal sorting complex required for transport (ESCRT) (Katzmann et al., 2002; Wollert and Hurley, 2010) prevents canonical Wnt signaling (Taelman et al., 2010). Because endolysosomal function is essential for Wnt signaling, we decided to investigate the effect of inhibitors of lysosomal function on Wnt signaling. Lee et al., 2010 recently reported that presenilin 1 (PS1), an intramembrane protease that is mutated in early-onset familial Alzheimer's disease (FAD), is required for proper autophagosome digestion. These authors found that acidification of lysosomes was impaired in PS1-deficient cells, and proposed a model in which PSs are required for lysosomal maturation. An extensive literature links autophagy defects with neurodegeneration (Nixon et al., 2008). Furthermore, toxic amyloid precursor protein (APP) peptides accumulate intracellularly specifically in MVBs in early-stage AD (Takahashi et al., 2002), and certain polymorphisms in the lysosomal protease cathepsin D increase the risk for AD (Nixon and Yang, 2011). Taken together, these observations motivated us to investigate whether PS deficiency might affect Wnt signaling through functional changes in the endolysosomal system.
In this study, we found that canonical Wnt signaling activity was significantly increased when lysosomal function was inhibited by chloroquine (CQ), a drug that raises lysosomal pH. Depletion of PS1 or PS2 also resulted in a significant increase of Wnt transcriptional activity in T cell factor (TCF)/β-catenin reporter gene assays. In both cases, lysosomal function was inhibited downstream of ILV formation. Indeed, the ESCRT machinery, which generates ILVs, and the small GTPase Rab7, which is involved in MVE/late-endosome formation (Bucci et al., 2000; Huotari and Helenius, 2011), were both required for Wnt transduction. In electron microscopy analyses, CQ-treated or PS-deficient cells displayed a striking accumulation of late-endosomal vacuoles containing ILVs. Immunofluorescence microscopy studies indicated that these MVEs were positive for Rab7 and the ILV membrane marker CD63 (Escola et al., 1998). The expanded late endosomes sequestered more GSK3/Wnt-receptor complexes compared with wild-type (WT) cells when the cells were treated with Wnt. Several GSK3 protein substrates, such as β-catenin, the previously developed GFP-GSK3-protein stability biosensor (Taelman et al., 2010), and the microtubule-associated protein Tau-GFP, became more stabilized by Wnt in PS1-depleted cells. The results presented here highlight the key role played by the endolysosomal pathway in Wnt signal transduction.
CQ Increases Wnt Signaling via MVEs
To investigate the role of the endolysosomal system in Wnt signaling, we used CQ, an antimalarial drug. CQ is a weak base that accumulates in lysosomes, causing their alkalinization and inhibiting the activity of acidic hydrolases (Wibo and Poole, 1974). Previous studies have described sensitive transcriptional reporters of Wnt activity in which multiple TCF binding sites (called SuperTopFlash and BAR reporters) activate transcription when β-catenin accumulates in the nucleus (Veeman et al., 2003; Biechele and Moon, 2008). As shown in Figures 1A and 1B, Wnt signaling was stimulated up to 7-fold by CQ treatment in HEK293T cells (brackets at left). Increases in Wnt signaling were also observed with the use of the lysosomal protease inhibitors leupeptin and E64 (increases of 2.6- and 2.1-fold, respectively; Figure S1A). Therefore, interference with lysosomal maturation and function potentiates Wnt signaling.
To test whether the amplification of Wnt signaling by CQ required the formation of ILVs in MVEs, we used small interfering RNA (siRNA) knockdown of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), also known as vacuolar protein sorting 27 (Vps27), which is required for early stages of ILV formation (Katzmann et al., 2002; Taelman et al., 2010). HRS/Vps27 was required for the stimulation of Wnt signaling by CQ (Figure 1A, right bracket). Vps4 (another ESCRT component required for ILV formation) and Rab7 (a protein required for late-endosomal maturation) were also required (Figure 1B, right bracket; data not shown).
To determine whether CQ causes the accumulation of MVEs, we examined 3T3 cells treated overnight (Figures 1C–1E) or L-cells treated with CQ for 6 hr, 1 hr, or 15 min (Figures 1F and 1G) by transmission electron microscopy. In both cell lines, CQ treatment caused a striking increase in autolysosomes containing accumulations of electron-dense cytoplasmic materials, such as aggregated proteins, as well as small ILVs ∼50 nm in diameter (which are characteristic of MVEs; Figures 1D and 1E). No double-membrane macroautophagic vacuoles were seen, even at early time points. However, the MVEs were clearly engaged in microautophagy (Sahu et al., 2011), because after only 15 min of CQ treatment, many showed large invaginations of the limiting membrane (Figure 1G, arrows) enclosing regions of cytoplasm containing fine electron-dense granules corresponding to ribosomes. After the limiting membrane is pinched off, these invaginations generate electron-dense deposits in late autolysosomes, which are then enveloped by single or multilaminar membranes depending on the stage of the individual autolysosomes (Figure 1E).
CQ treatment causes the rapid accumulation of endosomes marked in their outer membrane by Rab7 (Figures S1B–S1C″′ and Movie S1. CQ Treatment Rapidly Expands the Rab7-GFP-Positive Compartment, Related to Figure 1). These vacuoles correspond to MVEs because they colocalize with the tetraspanin protein CD63, a marker for ILV membranes (Figures S1D–S1E″; Escola et al., 1998). Flow cytometry confirmed that the CQ-treated cells had increased levels of Rab7 (by 25.2%) and CD63 (by 28.5%) antigens in the cell population as a whole (Figures 1H and 1I).
We conclude that the inhibition of lysosomal function caused by CQ does not prevent the formation of endosomal ILVs, but instead enhances it. The MVE expansion would enhance the sensitivity of cellular responses to Wnt, which are entirely dependent on the ESCRT machinery.
Dose-Dependent Response of Wnt Signaling to CQ and Bafilomycin
We next investigated the effects of inhibiting lysosomal function on Wnt signaling with different doses of CQ or the specific vacuolar ATPase (v-ATPase) inhibitor bafilomycin A (Figures 2A and 2B). The v-ATPase enzyme acidifies the entire endosomal pathway as vesicles traffic from the plasma membrane to lysosomes. We found that low concentrations of CQ or bafilomycin increased Wnt3a responses in LSL cells stably transfected with a TCF reporter, whereas at higher concentrations (>250 μM for CQ and >50 nM for bafilomycin) both drugs inhibited Wnt signaling, presumably by alkalinizing the early-endosomal compartment as well (Figures 2A and 2B). Because the generation of ILVs within the expanded endosomes can still proceed in the presence of CQ (Figure 1E), the accumulation of ILVs in MVEs may provide the basis for the increased sensitivity to Wnt signals after CQ treatment. At high CQ concentrations, the more-extensive alkalinization of the entire endolysosomal pathway may actually inhibit ILV formation or receptor activation. Our results agree with a previous study by Cruciat et al., 2010 showing that LRP6 phosphorylation requires acidification of early endosomes/signalosomes in HEK293T cells treated with high concentrations of bafilomycin that inhibit Wnt signaling. We now extend their findings by showing that at low doses v-ATPase inhibition increases Wnt signaling, presumably reflecting a higher requirement for the proton pump in the more-acidic late-endosomal compartment. These results highlight the link between Wnt signal transduction and membrane trafficking.
PS Deficiency Leads to Increased Sensitivity to Wnt Signals
A new function for PS in the maturation of v-ATPase was recently reported (Lee et al., 2010), motivating us to explore the impact of lysosomal malfunction caused by PS deficiency on Wnt signaling. We first confirmed that knock out or siRNA depletion of PS1 indeed generated less acidified endosomes, as was shown by reduced staining with LysoTracker which marks acidic organelles (Figures S2A–S2E′). Efficient knockdown of PS1 was demonstrated with an immunoblot using a Flag-tagged PS1 construct (Figure S2F). We then extended the observations of Lee et al., 2010 by showing that the fluid-phase endosomal compartment, measured by endocytosis of DextranRed (tetramethylrhodamine Dextran), was significantly expanded by PS1 knockdown with siRNA (Figures 2C and 2D). Flow cytometry confirmed the increase in DextranRed-accessible endosomes in the PS1-depleted cell population in a quantitative way (Figure 2E).
Endosomal expansion was accompanied by an increase in Wnt responsiveness in PS1-depleted HEK293T cells, particularly at higher Wnt3a doses (Figure 2F, bracket). The stimulation of Wnt signaling caused by PS1 siRNA-mediated depletion was entirely dependent on ILV formation in MVEs because, as shown in Figure 2G, HRS/Vps27 depletion strongly inhibited Wnt signaling in control siRNA or PS1 siRNA-treated HEK293T cells. We extended these findings to the Xenopus animal cap system (Taelman et al., 2010). We designed a PS1 morpholino oligo (MO) that reproducibly increased signaling by SuperTopFlash reporter in cells coinjected with xWnt8 mRNA (Figure 2H), and this effect was rescued by human PS1 coinjection (Figure S2G). This increase in Wnt signaling was blocked by HRS/Vps27 MO (Figure 2H, right bracket). We conclude that the ESCRT machinery is required for the effects of PS1 knockdown both in cultured cells and in the in vivo animal cap system.
We also tested whether PS2 affected Wnt signaling using siRNA. Depletion of PS1 or PS2 led to increased responsiveness in Wnt luciferase assays (Figure 2I), indicating that both proteins have functional overlap, at least in HEK293T and HeLa cells (see also Figures 3A–3D″ below). Although the increase in Wnt signaling by PS1 siRNA was higher than that by PS2 siRNA, both together had additive effects (Figure 2I). The effects of PS2 siRNA on Wnt signaling, as well as those of PS1, could be rescued by overexpression of the human PS1 gene (Figure S2H). Interestingly, FAD-associated PS1 mutations (M146V, A246E, and L392V) were much less effective at rescuing the Wnt signaling increased by PS1 depletion (Figure S2I). In contrast, PS1 mutations (D257A and D385A) in the aspartates required for proteolytic activity were as effective as WT PS1 in rescuing the effect on Wnt signaling (Figure S2I). This is in agreement with previous work showing that the effects on PSs on autolysosomes are independent of γ-secretase enzyme activity (Lee et al., 2010; Neely et al., 2011). The fact that FAD point mutations behave differently from protease-deficient PS1 could be relevant for disease.
Taken together, these data suggest that PS depletion increases the capacity of cells to respond to Wnt by promoting the generation of ESCRT-dependent ILVs in the endosomal pathway.
Late Endosomes Accumulate in PS1- and PS2-Deficient Cells
PS depletion had a dramatic impact on the endolysosomal system. In electron microscopy studies, depletion of PS1 or PS2, or the simultaneous depletion of both gene products, resulted in an increase in the number and size of single-membrane-bound vacuolar structures in HeLa cells (compare Figure 3A with Figures 3B–3D). These structures were autolysosomes and MVEs, as they contained a variable number of small and uniform ILVs (Figures 3B′–3D′) as well as electron-dense, undigested cytoplasmic material. Double-membrane macroautophagic vesicles were not observed. Some of the most striking MVEs are shown in Figures 3A″–3D″. Immunofluorescence analyses showed increased staining for the endosomal pathway markers Rab7 and LAMP1, as well as for the ILV marker CD63 when PS1 or PS1/2 were depleted with siRNA (Figure S3; data not shown). Flow cytometry showed increased levels of Rab7 (by 30.9%) and CD63 (by 35.7%) in the entire cell population after PS1 knockdown (Figures 3E and 3F).
We conclude that PS1 and PS2 play a fundamental role in endolysosomal biogenesis and function, and, as in the case of CQ, PS deficiency blocks the pathway downstream of ILV formation.
PS Depletion Leads to Increased Phospho-LRP6 and Nuclear β-Catenin upon Wnt Signaling
Given the expansion of the MVE compartment in PS1-depleted cells, we next asked whether trafficking of the activated Wnt coreceptor LRP6 was affected upon Wnt ligand binding. Phosphorylation of LRP6 marks the initial activation step in the Wnt signaling cascade (Niehrs and Shen, 2010). HEK293T cells stably expressing LRP6-GFP (Kategaya et al., 2009) were transfected with control or PS1 siRNAs and treated with high concentrations of Wnt3a overnight, and activation of LRP6 was detected with a phosphospecific anti-p-LRP6 antibody (Figures 4A–4F). Phospho-LRP6 immunostaining in vesicle-like cytoplasmic puncta (Bilic et al., 2007) was detected only in cells treated with Wnt (arrows), and was significantly higher in cells lacking PS1 (compare Figures 4D and 4B). Only a subset of cells displayed strong responses to Wnt, probably due to the cell-cycle dependence of Wnt signaling (Davidson et al., 2009; Niehrs and Acebron, 2012). Immunoblot analyses confirmed that PS1 knockdown caused a reproducible increase in LRP6 phosphorylation in the cell population as a whole, whereas total levels of nonphosphorylated LRP6-GFP were not affected (Figures 4E and 4E′). Accordingly, the ratio of pLRP6/LRP6-GFP was increased by Wnt in PS1-depleted cells (Figure 4F).
Accumulation of β-catenin in the nucleus constitutes one of the hallmarks of the Wnt signaling cascade. Nuclear accumulation of β-catenin was detectable only after Wnt treatment in HeLa cells (Figures 4G–4J′; arrows indicate the locations of individual nuclei). The increase in both nuclear and cytoplasmic β-catenin was highest in PS1-depleted cells treated with Wnt (Figure 4J). The increase in β-catenin levels was quantified in immunoblot analyses (Figures 4K and 4K′). A weak but significant increase in β-catenin was also observed in PS1-deficient cells even in the absence of the Wnt ligand (1.38 ± 0.08-fold increase over control; Figure 4K, lanes 1 and 3). However, this increase of β-catenin in PS1-depleted cells (see also Soriano et al., 2001, and Kang et al., 2002) did not generate a transcriptional Wnt signal (see Figures 2F, 2G, and 2I). This is explained by the observed accumulation in the cytoplasm (but not in the nucleus) in the absence of Wnt (compare Figures 4I and 4J).
We conclude that PS1 depletion causes increased levels of activated phospho-LRP6 receptor in cytoplasmic puncta (presumably corresponding to endosomal vesicles of the type shown in Figure 3) when cells are exposed to Wnt, and that this correlates with increased nuclear β-catenin accumulation.
GSK3 Translocates to Endosomes in PS-Deficient Cells upon Wnt signaling
To investigate whether the increase in Wnt signaling observed in CQ-treated or PS1-depleted cells could be explained by the GSK3 sequestration mechanism, we used HeLa cells transiently expressing GSK3-RFP. Overnight exposure of these cells to Wnt-conditioned medium led to a relocalization of GSK3-RFP into cytoplasmic puncta (compare Figures 5A and 5B), which could be visualized when cells were washed with the mild detergent saponin to reveal the endocytic compartment prior to fixation (see Extended Experimental Procedures). This relocalization of GSK3 to cytoplasmic puncta upon Wnt signaling was strongly enhanced when cells were treated with CQ (Figures 5C and S4A). These Wnt-induced GSK3 puncta colocalized with the late-endosome marker Rab7 (Figures 5B″ and 5C″).
We next used an activated form of Rab5 (Rab5QL-DsRed), which induces the formation of large MVBs (Wegner et al., 2010), to show that GSK3-GFP is translocated to endosomes. HeLa cells were transfected with either control or PS1 siRNAs, treated with Wnt3a-conditioned medium overnight, and treated with saponin before fixation. We observed an accumulation of GSK3-GFP puncta inside and in the periphery of Rab5QL MVBs, specifically when PS1-depleted cells were treated with Wnt (compare Figures 5F–5F″ with Figures 5G–G″; see also Figure S4B). We also found that Wnt treatment led to the translocation of GSK3-GFP to MVBs in control siRNA cells (Figures 5D–5D″ and 5E–5E″; see also Figure S4B), although to a lesser degree.
These results suggest that depletion of PS1 with siRNA or inhibition of lysosome function with CQ enhanced the translocation of GSK3 to late endosomes/MVBs that is normally triggered by Wnt.
GSK3 Protein Substrates Are Stabilized by Wnt in PS1-Depleted Cells
Because GSK3 is more efficiently sequestered in MVEs of PS1-deficient cells, we investigated whether GSK3 phosphorylation substrates would be more stable in these cells. Consistent with the GSK3 sequestration model, we observed that cytosolic GSK3 substrates such as β-catenin (Figure 4K) and a GFP-GSK3 protein stability biosensor primed by a mitogen-activated protein kinase (MAPK) site (Figure 6A; Taelman et al., 2010) were more efficiently stabilized in PS knockdown cells upon receiving a Wnt signal (Figure 6A). As a control, we used a mutated GFP biosensor construct in which the three GSK3 phosphorylation sites were mutated into alanines. We found that the GSK3-resistant biosensor became stabilized and its levels not affected by Wnt or PS1 siRNA (compare Figures 6B and 6B′ with Figures 6A and 6A′). This indicates that the effects of Wnt and PS1 siRNA are exerted at the level of the protein half-life via GSK3 sites.
Using the same experimental design, we found that transfected Tau-GFP (Kwan and Kirschner, 2005) was stabilized by Wnt treatment, and that this stabilization was more marked when PS1 was depleted (Figures 6D and 6D′). Tau is a microtubule-associated protein that plays an important role in AD and contains multiple GSK3 phosphorylation sites. When we measured Tau phosphorylation by GSK3 in immunoblots using an anti-phospho-Tau (T181) antibody, we found that Wnt signaling decreased phosphorylation in both control and PS1-depleted cells (Figures 6C and 6C′). These results support the hypothesis that Wnt signaling removes active GSK3 enzyme from the cytosol, decreasing the protein phosphorylations that target GSK3 protein substrates for degradation. The increased protein stabilization caused by Wnt in PS1-deficient cells, in particular in the case of Tau, could provide a new link among Wnt signaling, protein stabilization, and neurodegeneration.
Late Endosomes Are Required for Wnt Signaling
Recently, we proposed a model for sustained Wnt signaling through sequestration of GSK3 in MVEs (Taelman et al., 2010). In the study presented here, we investigated how Wnt signal transduction is affected when the function of the endolysosomal pathway is altered by inhibiting lysosomal function with CQ, low doses of bafilomycin A, the lysosomal protease inhibitors leupeptin or E64, or PS1 depletion. We found that lysosomal inhibition caused an expansion of the late-endosomal compartment, leading to a more efficient sequestration of GSK3 and the generation of an enhanced Wnt signal. The CQ effect had an absolute requirement for the ESCRT machinery, which is essential for the formation of ILVs in the endosomal compartment. The enhanced Wnt signaling caused by PS depletion required Hrs/Vps27 not only in cell culture experiments but also in vivo in explants from Xenopus PS1 morpholino-injected embryos. Figure 7 shows a proposed model for how lysosomal inhibition increases Wnt/GSK3 signaling and protein stabilization.
The antimalarial drug CQ, which alkalinizes lysosomes but still allows the formation of ILVs, caused a great expansion of the late-endosomal compartment. Not all signaling pathways were increased by CQ treatment: in the case of Sonic Hedgehog (Shh), signaling measured by a luciferase reporter was inhibited by CQ, whereas Hrs-siRNA increased signaling instead of inhibiting signaling as in the case of Wnt (data not shown). An increase in the fluid phase of the endosomal compartment (marked by endocytosed DextranRed) was observed after siRNA-mediated depletion of PS1. This gene has been linked to AD and was recently found to be critical for lysosomal maturation (Lee et al., 2010; Neely et al., 2011; Zhang et al., 2012). PS1 and PS2 are intramembrane proteases that form the catalytic core of the γ-secretase complex that cleaves the transmembrane domains of a large number of proteins. PSs are found in most cellular membranes, including in the ILVs of MVEs (De Strooper and Annaert, 2010). In the experiments reported here, lysosomal inhibition or PS depletion increased Wnt signaling in an ESCRT-dependent manner, but did not generate a signal in the absence of Wnt ligand. This effect could be due to an expanded surface of the limiting membrane of MVEs, or perhaps to changes in pH within the endosome that could increase the sensitivity of the endosomal membrane to undergo invagination to generate ILVs.
Recently published work by Li et al., 2012 described the formation of active Wnt receptor complexes containing most of the components of the β-catenin destruction complex, except for the E3 ligase βTrCP. In agreement with that report, we observed that βTrCP does not translocate into LRP6 endosomes, whereas β-catenin phosphorylated by GSK3 is sequestered inside these Wnt-specific endosomes (Taelman et al., 2010; our own unpublished results). These findings underscore the deep relationship between endosomal trafficking and the Wnt signaling pathway.
Depletion of PS Increases Wnt Signaling
PSs have been proposed to be involved in maturation of lysosomes through N-glycosylation of the V0a1 subunit of the v-ATPase complex needed for endosomal acidification (Lee et al., 2010). However, this mechanism was recently questioned (Zhang et al., 2012), and it was suggested instead that PSs may be involved in regulating the gene network associated with lysosomal biogenesis (Sardiello et al., 2009), and affect autophagy/lysosomal proteolysis independently of lysosomal acidification (Neely et al., 2011). Lee et al., 2010 observed a decrease of endosomal acidification in LysoTracker staining of PS1 knockout cells and siRNA knockdowns, and we were able to confirm their observations. We also found significant increases of MVE markers such as CD63, Rab7, and endocytosed DextranRed in cells depleted of PS1. The effects on lysosomal maturation were independent of γ-secretase activity (Lee et al., 2010; Neely et al., 2011; Figure S2I), excluding an effect on Notch or other γ-secretase-dependent pathways.
The enhancement of Wnt signaling by PS depletion described here was entirely dependent on ESCRT machinery (Figures 1A, 2G, and 2H). Thus, formation of ILVs of late endosomes is required for the enhanced effects of the Wnt ligand. Our observations differ from a report by Kang et al., 2002, who proposed that PS functions as an alternative scaffold for β-catenin degradation. Importantly, they found that loss of PS1 stabilized β-catenin in a Wnt-independent way, whereas in our experiments, addition of Wnt protein was required to trigger Wnt transcriptional activity in cell lines with low levels of endogenous Wnt signaling (Figures 2F, 2G, and 2I). We note, however, that in Xenopus explants PS1 MO did slightly increase β-catenin signaling (Figure 2H). In addition, PS1 siRNA caused a small but reproducible increase of cytoplasmic β-catenin levels in HeLa cells (Figures 4I and 4K'). We suggest that the reported Wnt-independent accumulation of β-catenin (Kang et al., 2002) may be explained in part by an endogenous autocrine Wnt signal that became enhanced though MVE expansion in PS1 knockout cells.
PS Depletion Increases Protein Stabilization by Wnt
Proteins that are normally degraded in lysosomes are taken up from the extracellular space by endocytosis, are membrane proteins, or are ingested from the cytosol via autophagy. Upon Wnt signaling, cells experience an additional effect, namely the endosomal sequestration of cytoplasmic GSK3, which is translocated into MVBs together with activated Wnt receptor complexes (Taelman et al., 2010; Dobrowolski and De Robertis, 2012). Here, we provide evidence that three cytosolic GSK3 substrates—the microtubule-associated protein Tau, β-catenin, and a GFP-GSK3 biosensor—were stabilized by Wnt signaling in PS1-depleted cells.
The experiments suggest a mechanism for Wnt signaling in PS-deficient cells. We propose that upon Wnt signaling, activated receptor complexes are internalized in endosomes and sequester GSK3 inside the expanded late endosomes together with the activated Wnt receptor complex, which is composed of multiple protein components phosphorylated by GSK3 (Figure 7). Once cytoplasmic levels of active GSK3 are sufficiently decreased, GSK3 protein substrates become less phosphorylated, their phosphodegron domains are not recognized by E3 polyubiquitin ligases, and GSK3 target proteins become stabilized.
This mechanism could play a role in AD, because PS deficiency increases the stability of multiple GSK3 substrates upon Wnt signaling. Some of these substrates, such as the microtubule-associated protein Tau, play crucial roles in the pathogenesis of AD. An extensive literature links Tau with GSK3 signaling (Hall et al., 2000; de Calignon et al., 2012). A role for Wnt signaling in prevalent neuropsychiatric diseases, including AD, schizophrenia, and autism, has been proposed (De Ferrari and Moon, 2006); however, studies performed to elucidate the function of Wnt in neurodegeneration have often generated contradictory results (summarized by Boonen et al., 2009). It is possible that Wnt signaling can result in different outcomes at different stages of AD progression. At early stages of AD, increased sequestration of GSK3 may stabilize many proteins, as shown here for Tau. However, chronic protein stabilization in PS1-deficient cells might eventually lead to failure of the endosomal pathway and Wnt signaling as the disease progresses. Once GSK3 ceases to be sequestered, elevated levels of cytosolic Tau might be more readily phosphorylated by GSK3, triggering AD pathology. It is possible that repeated Wnt stimulation through the course of a lifetime aging process, in combination with the accumulation of MVEs caused by defective trafficking, could result in increased protein stabilization and the eventual accumulation of the neurotoxic Amyloid β42 (Aβ42) peptide (De Strooper and Annaert, 2010).
In early stages of AD, Aβ42 and its intermediates localize to MVBs (Takahashi et al., 2002) and are released from neurons via exosomes (Rajendran et al., 2006) to form extracellular amyloid deposits (Vella et al., 2008). According to the amyloid hypothesis for the onset of AD (Selkoe, 1991; Hardy and Higgins, 1992; Hardy, 2009), extracellular amyloid plaque deposits would lead to altered kinase/phosphatase activities and hyperphosphorylation of Tau. Other recent studies suggested that phosphorylated Tau spreads in a prion-like manner through neuronal synaptic circuits (Liu et al., 2012; de Calignon et al., 2012; Kfoury et al., 2012). In addition, autophagy and lysosomal proteolysis defects have also been proposed to be involved in the pathogenesis of AD (Nixon et al., 2008; Lee et al., 2010).
The experiments presented here demonstrate that decreased PS expression causes an increase in Wnt signaling. The results also show that PS1 depletion causes a considerable expansion of the endosomal system, resulting from the inhibition of lysosomal function downstream of ILV formation. The connection between Wnt signaling and increased stability of GSK3 phosphorylation protein targets in PS-deficient cells could explain why AD neurodegeneration is most severe in the hippocampus, especially in its dentate gyrus, and cerebral pyramidal neurons (Brundin et al., 2010), because it is in these particular cells that physiological Wnt signaling in the adult brain is maximal (Maretto et al., 2003). We propose that during the aging process, defects in membrane trafficking may increase protein stabilization in the cytosol after repeated stimulation by Wnt signaling, contributing to the formation and deposition of intracellular protein aggregates. Whether the proposed mechanisms play a role in human pathology remains to be determined. In the meantime, the results presented here strengthen the intimate connection between Wnt signaling and the cell biology of intracellular membrane trafficking.
Experimental Procedures
Cell Culture and Knockdown Experiments
All cell lines used in this work (HeLa, 3T3, HEK293T, and L-cells) were cultured in Dulbecco's modified Eagle's medium (DMEM) complete medium containing 10% fetal bovine serum (FBS), 1% glutamine, and 1% Pen/Strep. For knockdown experiments in cultured cells, the siRNAs targeting human PS1, PS2, and HRS were ON-TARGETplus SMARTpool siRNAs from Thermo Scientific (L-004998, L-006018, and L-016835, respectively). The nontargeting control-siRNA pool (D-001810) was from the same company. For RNAi depletion experiments, cells were transfected with siRNAs 24 hr prior to transfections with DNA. For the rescue experiments, we introduced a full-length human PS1 gene (EHS1001-33034; Open Biosystems) into the pCS2+ expression plasmid and transfected 0.3 μg of this DNA into each well of a 12-well plate containing siRNA-pretreated cells. Knockdown experiments in Xenopus laevis were conducted using PS1 morpholino (sequence: TTCACTGGTGTCATTCATATTAGCT; Gene Tools, LLC) and Hrs morpholino (sequence: TGCCGCTTCCTCTTCCCATTGCGAA; Gene Tools, LLC).
Immunostaining, Flow Cytometry, and Western Blots
The following primary antibodies were used for immunostaining and flow cytometry: anti-Rab7 rabbit monoclonal (#9367; Cell Signaling) at 1:350, anti-CD63 mouse monoclonal (#556019; BD Biosciences) at 1:250, anti-β-catenin rabbit (H-102; SantaCruz) at 1:200, and anti-phospho-S1490-LRP6 rabbit (#2568; Cell Signaling) at 1:500.
For western blots, we used the following primary antibodies: anti-β-catenin (#C2206; Sigma) at 1:4000, anti-Flag monoclonal (#F1804; Sigma) at 1:1,500, anti-GFP (#A6455; Molecular Probes) at 1:500, anti-phospho-LRP6 (#2568; Cell Signaling) at 1:500, anti-phospho-Tau (T181, #5383; Cell Signaling) at 1:500, and anti-α-Tubulin monoclonal (#CP06; Calbiochem) at 1:1,500. Secondary antibodies coupled to Infrared Dyes (IRDye 680 and IRDye 800) at 1:3,000 (LI-COR) were used, and western blots were analyzed using a LI-COR Odyssey system.
The results of three or more independent experiments are given as the mean ± SEM. Statistical analyses were performed with Excel (Microsoft), applying the two-tailed t test as appropriate. Significant differences of means are indicated as ∗ ≤ 0.05, ∗∗ ≤ 0.01, and ∗∗∗ ≤ 0.005.
Additional Methods
Details regarding the methods used for luciferase assays, endolysosomal staining, in vivo time-lapse movies, and electron microscopy are provided in Extended Experimental Procedures.
Luciferase Assays, LysoTracker and DextranRed Staining, hPS1 Mutant Constructs, and Time-Lapse Movies
For luciferase assays, cells were incubated with control or Wnt3a conditioned medium overnight, washed, lysed and measured using the Dual Luciferase Renilla Reporter Assay kit (#E1960) in the Promega Glomax plate luminometer following manufacturer's instructions. For a single well of a standard 12 well plate cells were grown up to a 80% confluency and transfected with 0.2 μg of BAR-Luciferase, 0.02 μg of CMV Renilla, and 0.3 μg of constructs such as GSK3 biosensor or Tau-GFP supplemented with carrier PCS2 empty vector to a final DNA amount of 1 μg.
Wnt3a conditioned medium, as well as control conditioned medium, were harvested from stably transfected L-cells according to the ATCC protocol (Willert et al., 2003), except for using a lower 2% FBS concentration, which we found to be more reliable and active. For the experiments in Figure 4, Wnt conditioned medium was supplemented by the addition of 80 ng/ml of purified Wnt3a protein (PeproTech, NJ). CQ diphosphate (Sigma) was dissolved in H2O as a 100 mM stock solution and added to cell cultures at about 80% confluency. Transfections of DNA constructs were performed using BioT (Bioland); for siRNAs reverse transfections Lipofectamine 2000 (Invitrogen) was placed first into the plates and mixed with trypsinized cells. For visualization of the endocytic compartment, living HeLa cells were treated with 0.01% (weight/vol) saponin in PBS, gently shaking for 5 min at room temperature prior to fixation (Bishop and Woodman, 2000). Endosomal staining (Figure S4) was quantified using the ImageJ software and ITCN plugin that measures intensity maxima from images based on individual parameters (http://www.bioimage.ucsb.edu). For LysoTracker staining, living cells were incubated in pre-warmed culture medium containing diluted LysoTracker Red DND-99 reagent (1:1000, Invitrogen #L7528) for 1 to 3 min at 37°C (depending on the cell line), immediately and quickly washed twice with PBS and fixed with 4% PFA for 15 min at room temperature. Other procedures were as described in Taelman et al., 2010.
For DextranRed staining, HeLa cells were grown on clean glass coverslips, transfected with the appropriate siRNAs and incubated with 2 mg/ml dextran Tetramethylrhodamine (D1868, Invitrogen, NY) diluted in DMEM complete medium for 30 min. Subsequently, cells were washed, fixed with 4% Paraformaldehyde in PBS and mounted on glass slides for microscopic evaluation.
Stably transfected LSL-cells containing SuperTopFlash-Luciferase constructs (Blitzer and Nusse, 2006) and HEK293T cells stably expressing LRP6-GFP (Kategaya et al., 2009) fusion proteins were generously provided by Drs. Roel Nusse and Randall Moon, respectively.
Human Presenilin 1 (PSEN1, transcript variant 1) from an EST was subcloned, together with a C-terminal Flag-tag into the expression vector pCS2 for rescue experiments. The hypomorphic FAD mutations A246E, L392V and M146V (Lee et al., 2010; Boonen et al., 2009), and the aspartate protease-inactive mutants D257A and D385A (Lee et al., 2010) were introduced by site-directed mutagenesis using Quick-Change II XL kit from Stratagene.
For filming in vivo time-lapse movies with Rab7-GFP and CQ, transfected NIH/3T3 cells were transferred to Lab-Tek coverglass chambers (#155380, Nalge Nunc International, New York). Pictures were taken every 5 min using a Zeiss Observer .Z1, equipped with Apotome.2, fully automated-stage and Temp/CO2Module S, using a Colibri 488 LED or DIC optics with an EC Plan-Neofluar 40x/0.75 M27 objective. CQ was added just before acquisition started.
Electron Microscopy
Confluent cultures of 3T3 cells in 10 cm dishes treated with 50 μM CQ for the indicated times or of HeLa-cells depleted of PS1 and/or PS2 with siRNA were fixed by replacing the medium with fixative solution (2% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4). After incubation in cold fixative for one hour, the cells were scraped from the dishes using a Teflon scraper, collected in Eppendorf tubes and recovered by centrifugation for 10 min 10,000 rpm. Cell pellets remained under the fixative for additional 3 hr at RT and were carefully washed three times for 10 min each with 0.1 M Cacodylate buffer. The pellets were post fixed with 2% Osmium tetroxide and processed for embedding in epoxy resin LX 112. Thin sections were stained with Uranyl acetate and Lead citrate and examined by TEM in a JEOL EX 1200 electron microscope.
We thank Drs. R. Nusse for LSL cells, R. Moon for HEK293T LRP6-GFP cells and the SuperTopFlash and BAR Wnt reporter plasmids, M. Kirschner for xTau-GFP, B. van Deurs for Rab7-GFP constructs, and K. Ohmi for advice. Flow cytometry was performed in the UCLA Jonsson Comprehensive Cancer Center, the Center for AIDS Research Flow Cytometry Core Facility (which is supported by National Institutes of Health [NIH] awards CA-16042 and AI-28697), and the David Geffen School of Medicine at UCLA. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DO1429/1-1 to R.D. and VI574/1-1 to P.V.) and the NIH (HD21502-25). E.M.D.R. is an Investigator of the Howard Hughes Medical Institute.
Supplemental Information
- Movie S1. CQ Treatment Rapidly Expands the Rab7-GFP-Positive Compartment, Related to Figure 1 (MOV 1289 kb)
- A 240 min time-lapse movie of NIH/3T3 cells transfected with Rab7-GFP visualizes late endosomes in vivo. CQ treatment of the cells (movie in the top panel) showed a continuous increase of the Rab7-positive compartment. Note the rapid increase of Rab7-GFP fluorescence intensity and size of individual endosomes labeled with Rab7-GFP on their limiting membrane. Untreated control cells show no alteration of Rab7-GFP signal during the same time interval (movie in the lower panel). The acquisition rate was 12 frames per hour (49 frames in total), and the movie is played at four frames per second.
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Publication Information
Received: August 1, 2012
Revised: September 8, 2012
Accepted: September 24, 2012
Published: November 1, 2012

subset: commoncrawl


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In one year - Austin --> Boston --> London --> NYC. Adventurous eater. Cyclist. World traveler. Music fanatic. Free thinker. Wine drinker. Dog enthusiast. Lover of big cities and wide open spaces. Ambitious. Space and tech nerd. Appreciator of sarcasm and silliness alike. Butterfly of the social kind. Embracer of life.
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From ErfWiki
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Parson looks over at Wanda, in her red armor, sitting on a rock with the Arkenpliers on her knees. She has a cold look on her face. Nearby, two decrypted men stand in front of a huge rock, looking confused. Similarly, a gobwin stands perplexed at his situation in front of a stone outcropping. In the distance, more decrypted troops stand idly about.
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Wanda Firebaugh: I am loyal to Fate magic. I play what parts I must.
You do not know what I have done.
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DO WANT: Street Hawk Urban Assault Vehicle
Seen here demonstrating how Power Wheels are made, this is the The Shadow Hawk Street Hawk: coming soon to my driveway elevator garage. Okay maybe not. But only because I could never part with the Tercel.
The Street Hawk is the first vehicle with 46 inches of independent wheel travel without camber or caster compromise. The ride height or ground clearance is adjustable from zero to 44 inches while in motion. The Street Hawk has 1,100 horsepower, 1,805 foot pounds of torque and has a highway estimated 22mpg. The vehicle is all-wheel-drive with 40 inch tall, 15.5 inch wide tires and 22 inch wheels. The air suspended seats are hand crafter to the driver's specific proportions. From the driver's seat you can control the vehicles pitch, roll and overall ride height using D-pad controls beneath your fingertips.
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I want one. No, I NEED one. And they start at only $1.2 million. I'm gonna buy all of next year's production! In my dreams. And speaking of my dreams: I had one last night where I was making out with a werewolf (I blame Twilight). So yeah, what's that mean? And, completely unrelated, any idea why my dog's been hiding under the bed all day? Must be sick.
Hit the jump for a bunch more pictures.
For Sale: The most revolutionary and advanced vehicle EVER! [jameslist]
Go Go Gadget Hummer [gizmodo]
Thanks to PaperAK, Chris and arron, who are all holding out for Ripsaws. OMG I almost forgot about those!
So Porsche has decided to go forward and produce the Spyder 918 hybrid, which, up until now, was strictly conceptual. Did I mention it'll cost around $650,000? Because it will. Yeah, I just pre-ordered two. Didn't I, Hot Wheels? High rolla, HIGH ROLLA! A 3.4-liter V8 petr... / Continue →
The 2010 Cosmos 4RW V8 Muscle Bike sports four 17-inch wheels, a 250 cubic inch V8 producing 350 horsepower and an extra helping of deathtrap. Want one? Expect to spend $93K. Unless you're Batman, in which case it comes standard in the front end of a Tumbler. Unless he's be... / Continue →
We've seen chromed cars in the past, but quite frankly (can I call you Frankly?), those just weren't as shiny as this Bugatti Veyron. Maybe they went for the economy chrome job, but whatever the case, they lacked the mirror-like sheen of this summabitch. I mean, you could sno... / Continue →


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Three FORT scientists are presenting at the 40th Annual Symposium on Bat Research hosted by the North American Society for Bat Research (NASBR) October 27-30 in Denver, Colo. Ernest Valdez is presenting on "Diets of the sympatric Pacific sheath-tailed bat and Mariana swiftlet on Aguiguan, Mariana Islands." Paul Cryan will be presenting an overview of research needs for white-nose syndrome during a panel discussion on this devastating disease and a research poster entitled, "Evidence of mating readiness in certain bats killed by wind turbines." Laura Ellison is presenting a poster she coauthored with Dr. Cryan and retired USGS scientist Tom O'Shea, also related to white-nose syndrome in bats: "A Map of the Big Brown Bat Wing: 'Typical' Damage and Scarring in an Urban Maternity Population Prior to White-Nose Syndrome."
For more information contact: Ernest Valdez
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NOVA: Separating Twins
NOVA: Separating Twins
Thu, 03/07/2013 - 7:00pm
Witness the extraordinary surgery that will allow twin girls, born joined at the head, to live separate lives.
This is the incredible story of Trishna and Krishna, twin girls born joined at the head. Abandoned shortly after birth at an orphanage in Bangladesh, they had little chance of survival, until they were saved and taken to Australia by an aid worker. After two years battling for life, the twins are ready for a series of delicate operations, which will prepare them for the ultimate challenge: a marathon separation surgery that will allow them to live truly separate lives. Since the beginning, surgeons knew there was no guarantee of survival for either of the girls — but without surgery there was no hope at all. With exclusive access to this extraordinary human and medical drama, NOVA's cameras have been with Trishna and Krishna and their caregivers at each moment of their journey in NOVA: Separating Twins airing Thursday, March 7 at 7 p.m. on WXXI World (DT21.2/cable 524).


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When Carol Wall hired a neighbor's gardener to improve her long-neglected yard, she never imagined that the Kenyan immigrant would transform her outlook on life as well. In Mister Owita's Guide to Gardening, Wall reflects on what she learned from their special friendship.
Madhulika Sikka's new book, A Breast Cancer Alphabet, is here "for anyone who has been diagnosed with breast cancer and needs a companion."
Ape, chicken, cow, dog, pig, rat, sheep, snake, beast. Each of these words has a distinct connotation, none of them positive. The fact is, though, that no animal behavior can compete with the aggressive and destructive violence exhibited by humans on a regular basis. Animal advocate Jeffrey Moussaieff Masson has published numerous bestsellers about the rich emotional lives of animals. In his latest thought-provoking book, Beasts, Masson turns his attention to humans, posing the questions: who are the real beasts, why, and what can we do about it?
This month's Audio column has something for everyone: mystery lovers, readers of inspiring memoirs and seekers of exciting new voices in fiction.
BookPage Nonfiction Top Pick, March 2014
Walter Kirn has penned a number of imaginative novels, including Up in the Air and Thumbsucker, which were both made into movies. But nothing in the pages of those books could match the bizarre, real-life experiences Kirn relates in his new memoir, Blood Will Out.
The damp practically floats off the pages in Astoria, the sweeping tale of John Jacob Astor's attempt to settle the remote Pacific Northwest coast in 1810. Astor's vast wealth enabled him to send two expeditions: one over land and one by ship. His plan was to set up a fur trade, the first on this particularly harsh stretch of the West Coast. Whoever could settle the area would lay claim to a vast area rich with sea otter and beaver fur, salmon and other seafood.
It's hard to know whether to call Boyd Varty's Cathedral of the Wild a memoir, a true adventure story or a self-help book. All I know is that it made me cry with its hard-won truths about human and animal nature, distilled by Varty from his experiences living on Londolozi, the game reserve his family runs in South Africa.
Blake Bailey has written notable biographies of authors John Cheever and Richard Yates, both difficult and brilliant men. While he was sifting through their lives, he was also reflecting on his own. The Splendid Things We Planned is the resulting portrait, a story of mental illness and addiction and the difficult orbits they force upon the healthy. It's also a tribute to one family's best efforts and inevitable failings.
Debbie Stier faced a crisis. The oldest of her two children was approaching college age, and she hadn't saved for tuition. What's more, Ethan was, in her words: "a boy who was 'happy getting B's' and had gotten an awful lot of them." He was neither an honors student nor an extracurricular overachiever.
Dreaming of April in Paris? In How Paris Became Paris: The Invention of the Modern City, astute cultural observer Joan DeJean argues that Paris has been a modern, alluring city far longer than we usually imagine. Although we tend to think of 19th-century Paris as the bustling epitome of "la vie moderne," the roots of all we know and love about Paris today actually came into being in the 17th century.


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Homework Help
Better Students Ask More Questions.
How is Dr. Paul Farmer similar to philosopher Peter Singer in regards to social justice?
Topic: Mountains Beyond Mountains: The Quest of Dr. Paul Farmer, A Man Who Would Cure the World
How is Dr. Paul Farmer similar to philosopher Peter Singer in regards to social justice?
1 Answer | add yours
In Peter Singer's The Singer Solution to World Poverty, he proposes that humans, especially those who are living comfortable lives, should donate all of their money to the impoverished of the world instead of spending their money on luxuries. One could argue that Dr. Paul Farmer, a world-renowned infectious disease physician, lives out Singer's philosophy. While Farmer certainly has the credentials, skills, and contacts to live a life of luxury and fame, he chooses to devote almost all of his income and skills to the ill and impoverished people of struggling countries such as Haiti. Even when Farmer is in the United States working in Boston, he strives to help the addicted poor who are in his care. He spends personal money on basic necessities for his patients and operates a charity with the sole mission of providing medical care and necessities to the poor of the world. When Farmer is in Haiti, he maintains a Spartan existence so that all of his resources can be devoted to conquering TB and other medical issues in countries like Haiti.
Both men promote social justice through trying to provide equality for all people in regards to their access to basic necessities. Dr. Farmer is, arguably, the more admirable of the two men, because he consistently lives out the philosophy that he advocates.
Posted by scarletpimpernel on February 13, 2012 at 2:09 AM (Answer #1)
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Flight of the bloggers
Despite recent departures, Discover is rebuilding fast
By Curtis Brainard Dec 12, 2012 at 06:50 AM
Amidst a move from New York to Wisconsin, Discover magazine has lost some of its most popular science bloggers in... More
Who cares if it's true? - Modern-day newsrooms reconsider their values
What Is Russia Today? - The Kremlin's propaganda outlet has an identity crisis
And from the left…Fox News - There's more to Fox News' strategy of hiring liberals than creating a public boxing match
Why Skype isn't safe for journalists - Here are some alternatives for secure voice calls to use instead
Placing a bet on USA Today - Gannett has long felt the television model could translate into print. Now it's using its flagship paper to double down on that idea.
Email blasts from CJR writers and editors
Maybe everything when that name is "Satoshi Nakamoto"
Here's what happens when the readers choose the frontpage story
The numbers on the Daily Mail don't add up
Wanted: knitters to create jumpers for little penguins
Conservation group calls for donations of small knitted jumpers for birds who have been caught in oil spills
Stunning timelapse of Yosemite National Park
CJR's Guide to Online News Startups
ACEsTooHigh.com – Reporting on the science, education, and policy surrounding childhood trauma
Who Owns What
The Business of Digital Journalism
A report from the Columbia University Graduate School of Journalism
Questions and exercises for journalism students.


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The only makeup I wear with any regularity is lipstick, and six of the seven tubes I own have the following names:
- Target Red
- Red Passion No. 5
- In the Red
- Fire & Ice
- Hot Passion
- Ravishing Red
You got it. They are all shades of red.
Understandably, I was a sucker for Alyson Fox's new photography book "A Shade of Red: One Lipstick and One Hundred Women." Its subjects range from little girls to old women, of various ethnicities and many walks of life. The only thing they have in common is that they are wearing a shade of Revlon lipstick called "Certainly Red."
Fox says in her introduction that most of the women had never had a photographic portrait done. They were women she came upon, or were introduced to by others, or who knew someone she had photographed.
Of lipstick, she writes, "Sometimes, wearing it is a way to fit in … or a way to stand out. Sometimes it's a uniform or an armor. A gash. An incision. Sometimes it's an invitation … or a warning. Sometimes it's just something that goes with your dress. But whatever the reason, it is always transformative."
She's right. Red lipstick is hard to wear. It gets smeared on your teeth, is left behind on glasses and napkins (unless you use L'Oreal Infallible, which practically has to be removed with paint thinner) and, after you reach a certain age, runs into the wrinkles around your mouth. But you cannot wear it without being aware that you are wearing it.
This feeling of awareness is evident in the photographs, which are as varied as their subjects. One woman looks apprehensively out of her car window. Another sits on one end of her sofa, part of an elderly man visible on the other end. A little girl lies on the floor, cuddling her doll, an open Chinese parasol beside her. A middle-aged woman in a bikini squeezes water from her hair as she stands in a swimming pool. One holds a black cat, another a black dog. Another is a dead ringer for Nancy Reagan who, by the way, still manages to wear red lipstick at age 90.
Few of the women are smiling or laughing. Instead, they look pensive or dreamy, wary or resigned. One of the few exceptions is a woman in red ministerial robes, a broad red-rimmed grin on her face, arms outstretched as if she means to fly off to heaven.
After quickly flipping through the pages, I went back and studied the photos. What is that woman going to do with the knife blade she is holding to her chest? Who is the military man in the portrait hanging behind an elderly lady at her dressing table? Where is the woman clutching the red roses headed in her elevator car? What's going on with the woman sitting on a pile of chopped wood, scratching the ears of a pit bull? Why is that young woman standing in the corner of what appears to be a padded room?
My one criticism of the book is that the women's names appear in the back of the book, listed by page numbers, but there are no numbers on the pages. Maybe that was intentional.
A British army officer, Lt. Col. Mervin Willet Gonin, is quoted in the book, commenting on the liberation of the Bergen-Belsen concentration camp (where Anne Frank and her sister Margot died). He elevates the application of red lipstick from silly indulgence to vital sacrament:
"It was shortly after the British Red Cross arrived, though it may have no connection, that a very large quantity of lipstick arrived …Women lay in bed with no sheets and no nightie but with scarlet red lips, you saw them wandering around with nothing but a blanket over their shoulders, but with scarlet red lips … That lipstick started to give them back their humanity."
He got it. But he couldn't wear it.


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Address DME Elevators & Lifts 1717 Industrial Drive Montgomery, IL 60538
Phone 630.892.7400 Toll Free 800.821.4498 Fax 630.892.7401 Email firstname.lastname@example.com
Address DME Elevators & Lifts 9465 Counselors Row, Suite 241 Indianapolis, IN 46240
Phone 317.383.0623 Toll Free 800.646.5565 Email email@example.com
Wisconsin Office: Project locations within the Milwaukee and Madison Wisconsin metro areas, as well as the far Northeastern Illinois regions (Lake & McHenry Counties).
Address DME Elevators & Lifts 9016 58th Place, Suite 900 Kenosha, WI 53144
Phone 262.605.1205 Toll Free 800.893.6656 Email firstname.lastname@example.com
Privacy Statement: DME Elevators & Lifts is aware that the security of your private information from the use of our website is an important concern. We take the protection of your personal data very seriously and demonstrate a strong commitment to online privacy. In acknowledgment of that commitment, we have adopted a comprehensive policy to safeguard the privacy of all visitors to the DME Elevators & Lifts website.
Collection of Data: We store data on our servers for various security reasons. This data may include the name of your Internet service provider, click sources, the websites that you visit from our site and your Internet Protocol (IP) address. This information can be used to help diagnose problems with our server, to administer our website, to help identify you and to gather broad demographic information. The data we collect will only be used for the purpose of supplying you with the requested products or services, company marketing purposes or for other reasons for which you have given your consent, except where otherwise provided by law.
Security: DME Elevators & Lifts uses technical and organizational security precautions to protect your data from manipulation, loss, destruction or access by unauthorized persons.
Disclaimer: This site contains links to other sites. DME Elevators & Lifts is not responsible for the content or privacy practices of these sites.
Contact Us: Please contact us if you have any questions or concerns about our privacy policy.


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Quick Facts
- NAME: Anne of Austria
- OCCUPATION: Queen
- BIRTH DATE: September 22, 1601
- DEATH DATE: January 20, 1666
- PLACE OF BIRTH: Valladolid, Spain
- PLACE OF DEATH: Paris, France
- AKA: Anne of Austria, queen consort of France
- AKA: Queen Anne
- Full Name: Queen Anne of Austria
Best Known For
Anne of Austria, queen consort of France, was married to the 14-year-old Louis XIII and later mothered Louis XIV.
Think you know about Biography?
Answer questions and see how you rank against other players.
Play NowAnne of Austria. (2014). The Biography Channel website. Retrieved 05:54, Mar 09, 2014, from http://www.biography.com/people/anne-of-austria-9185816.
Anne of Austria. [Internet]. 2014. The Biography Channel website. Available from: http://www.biography.com/people/anne-of-austria-9185816 [Accessed 09 Mar 2014].
"Anne of Austria." 2014. The Biography Channel website. Mar 09 2014, 05:54 http://www.biography.com/people/anne-of-austria-9185816.
"Anne of Austria," The Biography Channel website, 2014, http://www.biography.com/people/anne-of-austria-9185816 [accessed Mar 09, 2014].
"Anne of Austria," The Biography Channel website, http://www.biography.com/people/anne-of-austria-9185816 (accessed Mar 09, 2014).
Anne of Austria [Internet]. The Biography Channel website; 2014 [cited 2014 Mar 09] Available from: http://www.biography.com/people/anne-of-austria-9185816.
Anne of Austria, http://www.biography.com/people/anne-of-austria-9185816 (last visited Mar 09, 2014).
Anne of Austria. The Biography Channel website. 2014. Available at: http://www.biography.com/people/anne-of-austria-9185816. Accessed Mar 09, 2014.
Born in Spain in 1601, Anne of Austria, queen consort of France, was married to the 14-year-old Louis XIII and later mothered Louis XIV. Anne had a difficult time with her husband and his chief minister, Cardinal de Richelieu. The tension between Anne and Richelieu worsened when the Cardinal declared war on Anne's brother and divided her loyalty. In 1651,
"God does not pay at the end of every week, but He pays."
Louis XIV took power and Anne lost her role as sole regent. She died in 1666 in Paris, France.
Early Life
Born on September 22, 1601, in Valladolid, Spain, Anne of Austria was the oldest daughter of King Phillip III and Margaret of Austria. She was especially close to her mother who helped her develop a deep devotion to her Roman Catholic faith. Sadly Anne lost her mother to illness when she was around the age of ten.
Barely in her teens, Anne became betrothed to France's King Louis XIII in 1612. The two royals had both been born in September 1601 within days of each other—a fact some took as a sign that the pair were destined to be together. Unfortunately, in reality, the union of these two teenagers was an exercise in political matchmaking, not romantic love.
Miserable Marriage
Leaving her native Spain, Anne of Austria wed King Louis XIII in November of 1615. The beautiful young queen struggled to fit in her new homeland. Her husband paid her little attention and his chief minister, Cardinal Richelieu, questioned her loyalty to France. Anne spent much of her time visiting churches and convents. She also enjoyed going to the theater.
Anne suffered a miscarriage in 1622 (one of several she experienced), and the loss of a potential heir strained her marriage further. King Louis XIII blamed her for the loss of the baby. A few years later, he became angry at Anne again for her flirtation with England's Duke of Buckingham. Anne found herself at odds with her husband in 1635 after France declared war on Spain. During this time, she corresponded with her brother—who had become King Phillip IV—on the sly. While she apparently never gave away any secrets, Anne was accused of treason by Cardinal Richelieu in 1637. She was eventually pardoned, but King Louis XIII remained suspicious of her for the rest of his life.
Queen Regent
After nearly twenty-two years of marriage, Anne of Austria finally delivered an heir to the French throne in 1638. Her son Louis was considered a stroke of tremendous good fortune. Anne was thirty-six years old when he was born. Without an heir, her position as a queen was tenuous. But her son's arrival helped cement her place as French royalty. For that reason, he was called Louis de Dieudonne, or "Gift of God." Two years later, Anne gave birth to another son, Phillip.
Anne of Austria was a doting parent and more hands-on with her children than most royals of the time. As the boys grew, Louis XIII's health began to fail. He died in 1643, leaving a provision for the country to be ruled by a regent counsel instead by Anne herself. She had the French parliament overturn Louis XIII's attempt to temper her power.
profile name: Anne of Austria profile occupation:
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Famous Queens
They are the famous women who were born into royalty, or found their way to it by marriage, and grew up to be the ecelectic empresses who have inspired countless stories, books, plays and films. As Great Britain's Queen Elizabeth II celebrates her Diamond Jubilee in 2012, a celebration of her 60 years on the throne, the queens who have long been admired—some for thousands of years—for their grace, public charm, dedication to philanthropy, finesse and fashion sense, come back into the spotlight. Explore the lives of notable queens such as Cleopatra VII, Queen Rania and Anna Ivanovna, from the time they were crowned, to their tragedies and milestones as rulers and consorts.
Famous Queens 31 people in this group
Famous People Named Anne
Take a look at famous people named Anne Sullivan, Anne Heche, and Anne Moody.
Famous People Named Anne 17 people in this group


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Enchanted Font by CBdO
Enchanted is a unique contemporary font that mimics the style of handwriting and brush scripts; yet is neither. Great for logos, captions and large bodies of text. Paragraphs set in Enchanted are easily read since the letters do not connect; aiding in its legibility. Enchanted contains seven stylistic alternates.
Please e-mail me updates and font release news!


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Pre-Teen Book Club
Date: Sat. February 09, 2013
Time: 3:00 pm
Harold Washington Library Center
400 S. State Street
About this event:
Join us to discuss From the Mixed-up Files of Mrs. Basil E. Frankweiler by E. L. Konigsburg while hanging out with friends, playing games and snacking at our pre-teen book club meeting. For children ages 9 to 12 years old. This program takes place in the Children's Program Room of the Thomas Hughes Children's Library.
Registration is required. Space is limited. Please call (312) 747-4200 to register.


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RFE: Conference Listings
This service is joint project of RFE and EconBiz.
- Hong Kong Institute for Monetary Research (HKIMR); The Hong Kong University of Science & Technology (HKUST)
Dates: | July 02-03, 2013 |
Description: | The HKIMR/HKUST Conference on Macroeconomics and International Finance |
Location: | Hong Kong, Two International Finance Centre, China |
Subject: | Programme: - The Great Recession: A Self-Fulfilling Global Panic Philippe Bacchetta - A bargaining theory of trade invoicing and prices Cedric Tille - Services and the Dynamics of Female Labor Supply Joseph Kaboski - A Microfounded Model of Chinese Capital Account Liberalization Paul Luk - Lemons with Naïveté Hui Tong - Housing Bubbles and the U.S. Macroeconomy Pengfei Wang - "Risky Investment with Limited Enforcement" Vincenzo Quadrin - The Euro Area Crisis: Need for a Supranational Fiscal Risk Sharing Mechanism? Davide Furceri - A Bayesian DSGE Model of Stock Market Bubbles and Business Cycles Jianjun Miao - Making Sense of Chinas Economic Policy Jim Riedel - Eurosclerosis and International Business Cycles Jenny Xu Capital Controls and Currency Wars Anton Korinek - Asset Allocation and Monetary Policy Harald Hau [gemäß den Informationen des Anbieters - according to site editor's information] |
JEL Code: | F |
URL: | http://www.hkimr.org/conferences_detail-id73 |


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Handling Fee Reminding: $2.50 handling fee will be charged for ALL items from the same series, product code beginning with AZU- if you order less than $60.00.
All natural fat free food. Beta carotene rich and high in fiber.
INGREDIENTS: Pumpkin.
Nutrition Facts
Serving Size: 1/2 cup (120 g)
Servings per Container: 3.5
Nutrient Amount % DV
Calories 50
Calories from Fat 0
Total Fat 0 g 0%
Saturated Fat 0 g 0%
Cholesterol 0 mg 0%
Sodium 0 mg 0%
Total Carbohydrate 10 g 2%
Dietary Fiber 4 g 16%
Sugars 4 g
Protein 1 g
Vitamin A 280%
Vitamin C 4%
Calcium 2%
Iron 8%
Condition | New |
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Florida Gulf Coast has easily put on the most entertaining show of the entire 2013 NCAA Tournament. For starters, they are the first ever 15th-seeded team to advance to the Sweet Sixteen. That alone was probably enough to inspire a fan to pay tribute to the team with a tattoo on his forearm, but there is so much more about this team to love.
Florida radio station B1039 shared the photo you see above on Wednesday, proclaiming it to be the first ever Dunk City tattoo. While I don't know if that's true, I can almost guarantee it won't be the last. For those of you who don't know, Dunk City refers to the nickname that has been given to FGCU because of the sick dunks like this they have thrown down throughout the tournament. Even the university itself has embraced the tag line.
And that's not all. Their head coach also has a former supermodel for a wife and a tremendous sense of humor about it. The team has captured the hearts of thousands of people across the country with their up-tempo style and unique postgame celebration antics. The more we discuss it, the more I'm going to want a Dunk City tattoo. Since I don't know how my wife would react to that, it's probably best if I stop now.
H/T Game On!
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Darren Sproles learned of trade plans via Twitter
Caddie Steve Williams still hasn't buried hatchet with Tiger
Report: Phil Jackson agreed to become a Knicks president
Bryce Harper explains how he got so ripped
Denver Broncos favorites to land Demarcus Ware?
Aqib Talib agrees to big deal with Broncos
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Concept of Risk
Updated : March 2011
European Centre on Geomorphological Hazards - CERG
Address: 3 rue de l'Argonne, 67083 STRASBOURG cedex France
Tel.: +33 3 90 24 09 43 / GSM. +33 6 87 82 97 09
Fax: + 33 3 90 24 09 00
E-mail: firstname.lastname@example.com
Web: http://www.cerg.eu/
The CERG is both a specialized research centre of the EUR-OPA Network and a non-governmental international association (defined on the basis of the French law 1901) with active and corresponding members. The CERG activities are supervised by an Executive Committee composed by a President, a Vice-President, three Advisory Members, an Executive Secretary and a Deputy Executive Secretary (members are elected by half each 3 years). Its role is to propose and validate the scientific and training activities of CERG. A General Assembly opened to all members is organised each year: in 2011, CERG includes 75 researchers (geologists, geomorphologists, engineering geologists, hydrologists, geotechnicians) from 23 laboratories and research centres of 14 European countries. Since 1988, the research programmes of the CERG are supported by the European Community and the Council of Europe and occasionally by national research programmes and other administrations.
President: Prof. Jordi Corominas (Technical University of Catalonia, Barcelona)
President of the Scientific Committee: Prof. Jordi Corominas (Technical University of Catalonia, Barcelona)
Director: Prof. Olivier Maquaire (University of Caen Basse-Normandie)
Deputy Executive Secretary: Dr Jean-Philippe Malet (University of Strasbourg)
Permanent staff of the Center: -
Development of multidisciplinary research in the field of geo-morphological and geo-hydrological hazards and dissemination of methodology and techniques through advanced training courses and guidebooks.
Promotion of scientific and technical co-operation between CERG European members and exterior individuals or organisations.
Training activities
· Several European Intensive Summer Courses (France –Martinique, -South Alps, Normandy, Italy, Austria, Portugal, Spain, etc.)
. Several Post-Graduate Training School have been organized in the framework of the FORM-OSE training activities:
- FORM-OSE - 3, September 2008 (Barcelona, Spain): Quantitative risk assessment and management
- FORM-OSE - 2, September 2006 (Bonn, Germany): Multi-RISK – Concepts to approach multi-hazards
- FORM-OSE - 1, September 2004 (Strasbourg, France): Living with hydro-geomorphological risks, from theory to practice
On-going activities:
· Implementation of the Be-Safe-Net website "Disaster Awareness with the use of the Internet".
· Preparation of "Post-Graduate Training School" in 2011.
- FORM-OSE - 4, June 2011 (Caen, France): Coastal hazard assessment and risk management
Research activities
· Seism and landslides: research on landslides triggered by earthquakes to improve their spatial prevision and their prevention. (research developed in North and Central Apennines, Portugal, etc.);
· Geomorphological mapping of the upper Tagliole valley (Modena Apennines, Northern Italy) ;
· Inventory of landslide risk assessment methodologies used in the EU-25;
· State of the art in multiple hazard and risk studies;
. Identification of thresholds for landslide crises, and implications for operative early warning systems.
· Landslide susceptibility mapping at the European scale
· Coastline at risk: methods for multi-hazard assessment
· Real-Time Management of Emergency Phase in the aftermath of Natural Disasters
. An update on multi-hazard risk assessments
. Applicability of Fibre Optic cables for landslide early warning systems.
Other activities
· Several research programmes have been prepared and financed through CERG's initiative: Epoch, Teslec, Newtech, Alarm, Ramsoil, Mountain-Risks, Changes, ChangingRisks, etc.;
· Several technical expertises in occasion of natural disasters in Europe: landslide in the Leusheni area (Moldova, 1997), debris flow threat to the city of Kvareli (Georgia, 2000), etc.;
· Several CERG members are involved in the Scientific Committee of national research programmes;
The complete list of publications is avalaible on the CERG website. Recent publications by CERG are :
· Malet, J.-P., Remaître, A, Bogaard, T. A. 2009. Landslide processes: from geomorphologic mapping to landslide modelling, Proceedings of the Conference, Strasbourg, 6-7 February 2009, CERG Editions, Strasbourg, 356 p.
· Castaldini, D, Gentili, B, Materazzi, M, Pambianchi, G. 2003. Geomorphological sensitivity and system response. Camerino - Modena Apennines (Italy) July 4-9, 2003. Proceedings of the Course, CERG Editions, Strasbourg, 175 p.
· Borgatti, L, Soldati, M. 2002. Relationships between man and the mountain environment in terms of geomorphological hazards and human impact in Europe. Dornbirn (Austria) 14 July 2002. IAG Symposium Proceedings, CERG Editions, Strasbourg, 109 p.
· Delahaye, D, Levoy, F, Maquaire, O. 2002. Geomorphology: from expert opinion to modelling, Proceedings of the Conference, Strasbourg, 26-27 April 2002, CERG Editions, Strasbourg, 381 p.
CERG has supported or participated to the organization of:
- International conference - Landslide processes: from geomorphologic mapping to landslide modelling, Strasbourg, 6-7 February 2009 - A tribute to Prof. Dr. Theo van Asch
- International conference - Geomorphology: from expert opinion to modelling, Strasbourg, 26-27 April 2002 - A tribute to Prof. Jean-Claude Flageollet
- Specific sessions are organized in international symposiums:
- Specific Session: EGU General Assembly, Vienna (Austria).
Back to the list of the Centres


text:
1. Case
2. Keyboard
This is a discussion on What accessory do you think you will need within the iPad Accessories for iPAD 1, 2 and 3 forums, part of the Apple iPad Discussions category; 1. Case 2. Keyboard...
case (for protection)
vga-cable (since i have no macbook (pro) to give presentations)
I've already asked my wife if she thinks she can make me a case. If she can't then the proppable Apple case looks real good. After that, it will be whatever adapter I need to play a video on my tv.
Mine will be Accessories
Apple Case
Bluetooth keyboard
LL Bean Sunwashed Canvas shoulder bag to carry it all in! (it has the old,distressed,
recession era, "I'm Poor" look, to deter muggers) LOL
I do like the look of the Hard Candy cases for some Bling, I'll get a few of those, since I'll be hiding the iPad in the distressed bag.
I'll probably get the Apple case to start with. Maybe the camera connection kit at some point, but not right away. If I can find a reasonably priced set of bluetooth stereo headphones that doesn't sound like crap, I'd like a pair of those too.
I will need some rugged hardshell case. Not sure which one I'll buy. Maybe something like [ame="http://www.amazon.com/OtterBox-Impact-Case-iPhone-Black/dp/B001KV2LK0/ref=pd_sim_dbs_cps_2"]this[/ame] if they scaled it up.
Looking forward to all the third party accessories to be out on April 3. A dock for sure, probably a bluetooth keyboard vrs. the dock keyboard.
I ordered the separate dock & keyboard.
I also ordered the apple case... it looks very functional...& I like the break-back design.
It also looks like you could dock it w/ the case still on... I am hoping the top flap will fold all the way back flat like a spiral notebook does.
The case does not seem to have a latch to keep it from opening up in a backpack.. that would be nice feature.
I would like to be able to personalize the case with my own artwork... & leave off or cover up that "please steal-me" apple logo (but thankfully the apple does not appear to glow)