First cleavage of the mouse embryo responds to change in egg shape at fertilization

Although mouse development is regulative, the cleavage pattern of the embryo is not random. The first cleavage tends to relate to the site of the previous meiosis. Sperm entry might provide a second cue, but evidence for and against this is indirect and has been debated. To resolve whether sperm entry position relates to the first cleavage, we have followed development from fertilization by time-lapse imaging. This directly showed cytokinesis passes close to the site of the previous meiosis and to both the sperm entry site and trajectory of the male pronucleus in a significant majority of eggs. We detected asymmetric distribution of Par6 protein in relation to the site of meiosis, but not sperm entry. Unexpectedly, we found the egg becomes flattened upon fertilization in an actin-mediated process. The sperm entry position tends to lie at one end of the short axis along which cleavage will pass. When we manipulated eggs to change their shape, this repositioned the cleavage plane such that eggs divided along their experimentally imposed short axis. Such manipulated eggs were able to develop to term, emphasizing the regulative nature of their development.     

A common open representation of mass spectrometry data and its application to proteomics research

A broad range of mass spectrometers are used in mass spectrometry (MS)-based proteomics research. Each type of instrument possesses a unique design, data system and performance specifications, resulting in strengths and weaknesses for different types of experiments. Unfortunately, the native binary data formats produced by each type of mass spectrometer also differ and are usually proprietary. The diverse, nontransparent nature of the data structure complicates the integration of new instruments into preexisting infrastructure, impedes the analysis, exchange, comparison and publication of results from different experiments and laboratories, and prevents the bioinformatics community from accessing data sets required for software development. Here, we introduce the 'mzXML' format, an open, generic XML (extensible markup language) representation of MS data. We have also developed an accompanying suite of supporting programs. We expect that this format will facilitate data management, interpretation and dissemination in proteomics research.     

Evidence for placental transfer of lipids during gestation in the viviparous lizard, Pseudemoia entrecasteauxii

During gestation in the viviparous lizard Pseudemoia entrecasteauxii, the fetus obtains nutrients from two sources: uptake of yolk components from the retained egg (lecithotrophy) and transfer of nutrients from the maternal circulation via the placenta (placentotrophy). Although net placentotrophy in this species is indicated by the observation that the neonate contains 1.7 times more dry matter than the egg, the placental transfer of lipid has not been previously demonstrated. Lipid analysis was performed on newly ovulated eggs and on neonates. The weight of total lipid per neonate (8.2+/-0.5 mg) is significantly (P=0.049) greater than that in the egg (6.8+/-0.4 mg), indicating that the placenta must contribute some lipid to the fetus. On the assumption that 50% of the lipid delivered to the fetus from either source is oxidized for energy, it is calculated that the placenta accounts for 58.5% of the fetal lipid requirements, with the remaining 41.5% being derived from the egg. The fatty acid compositions of the triacylglycerol and phospholipid recovered in the neonatal tissue differ substantially from those of the egg. In particular, the proportions of 18:2n-6 and 18:3n-3 are far lower in the neonatal lipids compared with the egg lipids. On the other hand, the proportion of 22:6n-3 in the phospholipid of the neonate is six times higher than in the phospholipid of the egg. The absolute amount (mg) of 22:6n-3 recovered in the total lipid of the neonate is 3.8 times greater than the amount initially present in the egg. By comparison, the amount of total fatty acid in neonatal lipid is 1.2 times greater than the amount in the egg. Thus, there is a preferential use of 22:6n-3 for tissue phospholipid synthesis during development. We conclude that there is net transfer of fatty acids across the placenta to the fetus of P. entrecasteauxii and a high degree of selectivity in the use of the various fatty acids for fetal tissue lipid synthesis.     

Xenopus U8 snoRNA binding protein is a conserved nuclear decapping enzyme

U8 snoRNP is required for accumulation of mature 5.8S and 28S rRNA in vertebrates. We are identifying proteins that bind U8 RNA with high specificity to understand how U8 functions in ribosome biogenesis. Here, we characterize a Xenopus 29 kDa protein (X29), which we previously showed binds U8 RNA with high affinity. X29 and putative homologs in other vertebrates contain a NUDIX domain found in MutT and other nucleotide diphosphatases. Recombinant X29 protein has diphosphatase activity that removes m(7)G and m(227)G caps from U8 and other RNAs in vitro; the putative 29 kDa human homolog also displays this decapping activity. X29 is primarily nucleolar in Xenopus tissue culture cells. We propose that X29 is a member of a conserved family of nuclear decapping proteins that function in regulating the level of U8 snoRNA and other nuclear RNAs with methylated caps.     

During apoptosis bcl-2 changes membrane topology at both the endoplasmic reticulum and mitochondria

In healthy cells the antiapoptotic protein Bcl-2 adopts a topology typical of tail-anchored proteins with only the hydrophobic carboxyl terminus inserted into the membrane, as shown by labeling cell lysates with a membrane-impermeant sulfhydryl-specific reagent. Induction of apoptosis in cells triggered a change in the conformation of Bcl-2 such that cysteine 158 near the base of helix 5 inserted into the lipid bilayer of both endoplasmic reticulum and mitochondria where it was protected from labeling. Addition of a peptide corresponding to the BH3 domain of the proapoptotic protein Bim to cell lysates triggered a similar conformational change in Bcl-2, demonstrating that preexisting, membrane-bound Bcl-2 proteins change topology.     

Crystal structure of the BstDEAD N-terminal domain: a novel DEAD protein from Bacillus stearothermophilus

Most cellular processes requiring RNA structure rearrangement necessitate the action of Asp-Glu-Ala-Asp (DEAD) proteins. Members of the family, named originally for the conserved DEAD amino acid sequence, are thought to disrupt RNA structure and facilitate its rearrangement by unwinding short stretches of duplex RNA. BstDEAD is a novel 436 amino acid representative of the DEAD protein family from Bacillus stearothermophilus that contains all eight conserved motifs found in DEAD proteins and is homologous with other members of the family. Here, we describe the 1.85 A resolution structure of the N-terminal domain (residues 1-211) of BstDEAD (BstDEAD-NT). Similar to the corresponding domains of related helicases, BstDEAD-NT adopts a parallel alpha/beta structure with RecA-like topology. In general, the conserved motifs superimpose on closely related DEAD proteins and on more distantly related helicases such as RecA. This affirms the current belief that the core helicase domains, responsible for mechanistic activity, are structurally similar in DEAD proteins. In contrast, however, the so-called Walker A P-loop, which binds the beta- and gamma-phosphates of ATP, adopts a rarely seen "closed" conformation that would sterically block ATP binding. The closed conformation may be indicative of a general regulatory feature among DEAD proteins (and RNA helicases) that differs from that used by DNA helicases. BstDEAD also contains a unique extension of approximately 60 residues at the C terminus that is highly basic, suggesting that it might bind nucleic acids and, in so doing, confer specificity to the helicase activity of the core region.     

Continual expression of Rab5(Q79L) causes a ligand-independent EGFR internalization and diminishes EGFR activity

The amount of cell-surface Epidermal Growth Factor Receptor (EGFR) available to secreted ligand (EGF) dictates a cell's ability to mediate cell proliferation, differentiation or migration. Multiple factors regulate EGFR cell-surface expression including the rates of protein synthesis and protein degradation, and the endocytic trafficking of both stimulated and unstimulated EGFR. Rab5 is a 25 kDa protein that is localized to the plasma membrane and the early endosome. Its exact molecular function, however, remains controversial. We have used stable and transient expression systems in HeLa cells to examine the consequence of continual, overexpression of wild-type and activated mutants of rab5 on EGFR localization and signaling. Continual expression of constitutively activated mutants of rab5 causes a ligand-independent redistribution of EGFRs into intracellular vesicles that can not be blocked with an antagonistic antibody. The net result is a decrease in the level of cell-surface EGFRs available for ligand stimulation. Thus, rab5 activation regulates EGFR signaling by facilitating the internalization of the unliganded EGFR.     

Differential use of two AP-3-mediated pathways by lysosomal membrane proteins

The adaptor protein complex AP-3 is involved in the sorting of lysosomal membrane proteins to late endosomes/lysosomes. It is unclear whether AP-3-containing vesicles form at the trans-Golgi network (TGN) or early endosomes. We have compared the trafficking routes of endolyn/CD164 and 'typical' lysosomal membrane glycoproteins (lgp120/lamp-1 and CD63/lamp-3) containing cytosolic YXXPhi-targeting motifs preceded by asparagine and glycine, respectively. Endolyn, which has a NYHTL-motif, is concentrated in lysosomes, but also occurs in endosomes and at the cell surface. We observed predominant interaction of the NYHTL-motif with the mu-subunits of AP-3 in the yeast two-hybrid system. Endolyn was mislocalized to the cell surface in AP-3-deficient pearl cells, confirming a major role of AP-3 in endolyn traffic. However, lysosomal delivery of endolyn (or a NYHTL-reporter), but not GYXXPhi-containing proteins, was practically abolished when AP-2-mediated endocytosis or traffic from early to late endosomes was inhibited in NRK and 3T3 cells. This indicates that endolyn is mostly transported along the indirect lysosomal pathway (via the cell surface), rather than directly from the TGN to late endosomes/lysosomes. Our results suggest that AP-3 mediates lysosomal sorting of some membrane proteins in early endosomes in addition to sorting of proteins with intrinsically strong AP-3-interacting lysosomal targeting motifs at the TGN.