␥-Secretase facilitates the regulated intramembrane proteolysis of select type I membrane proteins that play diverse physiological roles in multiple cell types and tissue. In this study, we used biochemical approaches to examine the distribution of amyloid precursor protein (APP) and several additional ␥-secretase substrates in membrane microdomains. We report that APP C-terminal fragments (CTFs) and ␥-secretase reside in Lubrol WX detergent-insoluble membranes (DIM) of cultured cells and adult mouse brain. APP CTFs that accumulate in cells lacking ␥-secretase activity preferentially associate with DIM. Cholesterol depletion and magnetic immunoisolation studies indicate recruitment of APP CTFs into cholesterol-and sphingolipid-rich lipid rafts, and co-residence of APP CTFs, PS1, and syntaxin 6 in DIM patches derived from the trans-Golgi network. Photoaffinity cross-linking studies provided evidence for the preponderance of active ␥-secretase in lipid rafts of cultured cells and adult brain. Remarkably, unlike the case of APP, CTFs derived from Notch1, Jagged2, deleted in colorectal cancer (DCC), and N-cadherin remain largely detergent-soluble, indicative of their spatial segregation in non-raft domains. In embryonic brain, the majority of PS1 and nicastrin is present in Lubrol WXsoluble membranes, wherein the CTFs derived from APP, Notch1, DCC, and N-cadherin also reside. We suggest that ␥-secretase residence in non-raft membranes facilitates proteolysis of diverse substrates during embryonic development but that the translocation of ␥-secretase to lipid rafts in adults ensures processing of certain substrates, including APP CTFs, while limiting processing of other potential substrates. Sequential processing of amyloid precursor protein (APP)1 by ␤-and ␥-secretases releases the 39 -42-amino acid-long ␤-amyloid (A␤) peptides, which accumulate in the brains of aged individuals and patients with Alzheimer disease (AD) (1). The major ␤-secretase in neurons is an aspartyl protease termed BACE-1, which cleaves APP within the luminal domain, generating the N terminus of A␤ (2). The C terminus of A␤ is generated by intramembranous cleavage of APP C-terminal fragments (CTFs) by ␥-secretase, a multiprotein complex made of four essential components, presenilin (PS) 1 (or PS2), nicastrin, PEN-2, and APH-1 (3). Mutations in PSEN1 and PSEN2, encoding multipass membrane proteins PS1 and PS2, respectively, co-segregate with the majority of cases of autosomal dominant familial early-onset AD (4). Familial AD-linked PS1 and PS2 variants elevate the production of highly fibrillogenic A␤42 peptides (1). A role for PS1 in Notch function was first discovered in Caenorhabditis elegans screens and involves intramembranous cleavage of the Notch receptor, analogous to APP processing (5, 6). Nicastrin is a type I membrane protein independently identified as a novel component of the GLP-1/ Notch signaling pathway in C. elegans early embryos and in the biochemical characterization of proteins that interacted with PS1 (7,8). Multitransmembra...

Assessing how natural environmental drivers affect biodiversity underpins our understanding of the relationships between complex biotic and ecological factors in natural ecosystems. Of all ecosystems, anthropogenically important estuaries represent a ‘melting pot' of environmental stressors, typified by extreme salinity variations and associated biological complexity. Although existing models attempt to predict macroorganismal diversity over estuarine salinity gradients, attempts to model microbial biodiversity are limited for eukaryotes. Although diatoms commonly feature as bioindicator species, additional microbial eukaryotes represent a huge resource for assessing ecosystem health. Of these, meiofaunal communities may represent the optimal compromise between functional diversity that can be assessed using morphology and phenotype–environment interactions as compared with smaller life fractions. Here, using 454 Roche sequencing of the 18S nSSU barcode we investigate which of the local natural drivers are most strongly associated with microbial metazoan and sampled protist diversity across the full salinity gradient of the estuarine ecosystem. In order to investigate potential variation at the ecosystem scale, we compare two geographically proximate estuaries (Thames and Mersey, UK) with contrasting histories of anthropogenic stress. The data show that although community turnover is likely to be predictable, taxa are likely to respond to different environmental drivers and, in particular, hydrodynamics, salinity range and granulometry, according to varied life-history characteristics. At the ecosystem level, communities exhibited patterns of estuary-specific similarity within different salinity range habitats, highlighting the environmental sequencing biomonitoring potential of meiofauna, dispersal effects or both.

Epigenetic changes are among the most common alterations observed in cancer cells, yet the mechanism by which cancer cells acquire and maintain abnormal DNA methylation patterns is not understood. Cancer cells have an altered distribution of DNA methylation and express aberrant DNA methyltransferase 3B transcripts, which encode truncated proteins, some of which lack the COOH-terminal catalytic domain. To test if a truncated DNMT3B isoform disrupts DNA methylation in vivo, we constructed two lines of transgenic mice expressing DNMT3B7, a truncated DNMT3B isoform commonly found in cancer cells. DNMT3B7 transgenic mice exhibit altered embryonic development, including lymphopenia, craniofacial abnormalities, and cardiac defects, similar to Dnmt3b-deficient animals, but rarely develop cancer. However, when DNMT3B7 transgenic mice are bred with Eμ-Myc transgenic mice, which model aggressive B-cell lymphoma, DNMT3B7 expression increases the frequency of mediastinal lymphomas in Eμ-Myc animals. Eμ-Myc/DNMT3B7 mediastinal lymphomas have more chromosomal rearrangements, increased global DNA methylation levels, and more locusspecific perturbations in DNA methylation patterns compared with Eμ-Myc lymphomas. These data represent the first in vivo modeling of cancer-associated DNA methylation changes and suggest that truncated DNMT3B isoforms contribute to the redistribution of DNA methylation characterizing virtually every human tumor.

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