Trim17, novel E3 ubiquitin-ligase, initiates neuronal apoptosis

Accumulating data indicate that the ubiquitin-proteasome system controls apoptosis by regulating the level and the function of key regulatory proteins. In this study, we identified Trim17, a member of the TRIM/RBCC protein family, as one of the critical E3 ubiquitin ligases involved in the control of neuronal apoptosis upstream of mitochondria. We show that expression of Trim17 is increased both at the mRNA and protein level in several in vitro models of transcription-dependent neuronal apoptosis. Expression of Trim17 is controlled by the PI3K/Akt/GSK3 pathway in cerebellar granule neurons (CGN). Moreover, the Trim17 protein is expressed in vivo, in apoptotic neurons that naturally die during post-natal cerebellar development. Overexpression of active Trim17 in primary CGN was sufficient to induce the intrinsic pathway of apoptosis in survival conditions. This pro-apoptotic effect was abolished in Bax(-/-) neurons and depended on the E3 activity of Trim17 conferred by its RING domain. Furthermore, knock-down of endogenous Trim17 and overexpression of dominant-negative mutants of Trim17 blocked trophic factor withdrawal-induced apoptosis both in CGN and in sympathetic neurons. Collectively, our data are the first to assign a cellular function to Trim17 by showing that its E3 activity is both necessary and sufficient for the initiation of neuronal apoptosis. Cell Death and Differentiation (2010) 17, 1928-1941; doi: 10.1038/cdd.2010.73; published online 18 June 201

Embryonic and adult isoforms of XLAP2 form microdomains associated with chromatin and the nuclear envelope

Laminin-associated polypeptide 2 (LAP2) proteins are alternatively spliced products of a single gene; they belong to the LEM domain family and, in mammals, locate to the nuclear envelope (NE) and nuclear lamina. Isoforms lacking the transmembrane domain also locate to the nucleoplasm. We used new specific antibodies against the N-terminal domain of Xenopus LAP2 to perform immunoprecipitation, identification and localization studies during Xenopus development. By immunoprecipitation and mass spectrometry (LC/MS/MS), we identified the embryonic isoform XLAP2γ, which was downregulated during development similarly to XLAP2ω. Embryonic isoforms XLAP2ω and XLAP2γ were located in close association with chromatin up to the blastula stage. Later in development, both embryonic isoforms and the adult isoform XLAP2β were localized in a similar way at the NE. All isoforms colocalized with lamin B2/B3 during development, whereas XLAP2β was colocalized with lamin B2 and apparently with the F/G repeat nucleoporins throughout the cell cycle in adult tissues and culture cells. XLAP2β was localized in clusters on chromatin, both at the NE and inside the nucleus. Embryonic isoforms were also localized in clusters at the NE of oocytes. Our results suggest that XLAP2 isoforms participate in the maintenance and anchoring of chromatin domains to the NE and in the formation of lamin B microdomains

The Different Function of Single Phosphorylation Sites of Drosophila melanogaster Lamin Dm and Lamin C

Lamins' functions are regulated by phosphorylation at specific sites but our understanding of the role of such modifications is practically limited to the function of cdc 2 (cdk1) kinase sites in depolymerization of the nuclear lamina during mitosis. In our study we used Drosophila lamin Dm (B-type) to examine the function of particular phosphorylation sites using pseudophosphorylated mutants mimicking single phosphorylation at experimentally confirmed in vivo phosphosites (S25E, S45E, T435E, S595E). We also analyzed lamin C (A-type) and its mutant S37E representing the N-terminal cdc2 (mitotic) site as well as lamin Dm R64H mutant as a control, non-polymerizing lamin. In the polymerization assay we could observe different effects of N-terminal cdc2 site pseudophosphorylation on A- and B-type lamins: lamin Dm S45E mutant was insoluble, in contrast to lamin C S37E. Lamin Dm T435E (C-terminal cdc2 site) and R64H were soluble in vitro. We also confirmed that none of the single phosphorylation site modifications affected the chromatin binding of lamin Dm, in contrast to the lamin C N-terminal cdc2 site. In vivo, all lamin Dm mutants were incorporated efficiently into the nuclear lamina in transfected Drosophila S2 and HeLa cells, although significant amounts of S45E and T435E were also located in cytoplasm. When farnesylation incompetent mutants were expressed in HeLa cells, lamin Dm T435E was cytoplasmic and showed higher mobility in FRAP assay

Spatially Explicit Analysis of Metal Transfer to Biota: Influence of Soil Contamination and Landscape

Concepts and developments for a new field in ecotoxicology, referred to as “landscape ecotoxicology,” were proposed in the 1990s; however, to date, few studies have been developed in this emergent field. In fact, there is a strong interest in developing this area, both for renewing the concepts and tools used in ecotoxicology as well as for responding to practical issues, such as risk assessment. The aim of this study was to investigate the spatial heterogeneity of metal bioaccumulation in animals in order to identify the role of spatially explicit factors, such as landscape as well as total and extractable metal concentrations in soils. Over a smelter-impacted area, we studied the accumulation of trace metals (TMs: Cd, Pb and Zn) in invertebrates (the grove snail Cepaea sp and the glass snail Oxychilus draparnaudi) and vertebrates (the bank vole Myodes glareolus and the greater white-toothed shrew Crocidura russula). Total and CaCl2-extractable concentrations of TMs were measured in soils from woody patches where the animals were captured. TM concentrations in animals exhibited a high spatial heterogeneity. They increased with soil pollution and were better explained by total rather than CaCl2-extractable TM concentrations, except in Cepaea sp. TM levels in animals and their variations along the pollution gradient were modulated by the landscape, and this influence was species and metal specific. Median soil metal concentrations (predicted by universal kriging) were calculated in buffers of increasing size and were related to bioaccumulation. The spatial scale at which TM concentrations in animals and soils showed the strongest correlations varied between metals, species and landscapes. The potential underlying mechanisms of landscape influence (community functioning, behaviour, etc.) are discussed. Present results highlight the need for the further development of landscape ecotoxicology and multi-scale approaches, which would enhance our understanding of pollutant transfer and effects in ecosystems

Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability

Xenopus LAP2β protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2β fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2β antibody. Knockdown of the XLAP2β protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis