Nuclear envelope remnants: fluid membranes enriched in sterols and polyphosphoinositides.

BACKGROUND: The cytoplasm of eukaryotic cells is a highly dynamic compartment where membranes readily undergo fission and fusion to reorganize the cytoplasmic architecture, and to import, export and transport various cargos within the cell. The double membrane of the nuclear envelope that surrounds the nucleus, segregates the chromosomes from cytoplasm and regulates nucleocytoplasmic transport through pores. Many details of its formation are still unclear. At fertilization the sperm devoid of nuclear envelope pores enters the egg. Although most of the sperm nuclear envelope disassembles, remnants of the envelope at the acrosomal and centriolar fossae do not and are subsequently incorporated into the newly forming male pronuclear envelope. Remnants are conserved from annelid to mammalian sperm. METHODOLOGY/PRINCIPAL FINDINGS: Using lipid mass spectrometry and a new application of deuterium solid-state NMR spectroscopy we have characterized the lipid composition and membrane dynamics of the sperm nuclear envelope remnants in isolated sperm nuclei. CONCLUSIONS/SIGNIFICANCE: We report nuclear envelope remnants are relatively fluid membranes rich in sterols, devoid of sphingomyelin, and highly enriched in polyphosphoinositides and polyunsaturated phospholipids. The localization of the polybasic effector domain of MARCKS illustrates the non-nuclear aspect of the polyphosphoinositides. Based on their atypical biophysical characteristics and phospholipid composition, we suggest a possible role for nuclear envelope remnants in membrane fusion leading to nuclear envelope assembly.

Tubulovesicular structures within vesicular stomatitis virus G protein-pseudotyped lentiviral vector preparations carry DNA and stimulate antiviral responses via Toll-like receptor 9.

Recombinant lentiviral vectors (LVs) are commonly used as research tools and are being tested in the clinic as delivery agents for gene therapy. Here, we show that Vesicular stomatitis virus G protein (VSV-G)-pseudotyped LV preparations produced by transient transfection are heavily contaminated with tubulovesicular structures (TVS) of cellular origin, which carry nucleic acids, including the DNA plasmids originally used for LV generation. The DNA carried by TVS can act as a stimulus for innate antiviral responses, triggering Toll-like receptor 9 and inducing alpha/beta interferon production by plasmacytoid dendritic cells (pDC). Removal of TVS markedly reduces the ability of VSV-G-pseudotyped LV preparations to activate pDC. Conversely, virus-free TVS are sufficient to stimulate pDC and act as potent adjuvants in vivo, eliciting T- and B-cell responses to coadministered proteins. These results highlight the role of by-products of virus production in determining the immunostimulatory properties of recombinant virus preparations and suggest possible strategies for diminishing responses to LVs in gene therapy and in research use.

Stomach development is dependent on fibroblast growth factor 10/fibroblast growth factor receptor 2b-mediated signaling.

BACKGROUND & AIMS: Fibroblast growth factors (Fgfs) and their receptors (Fgfrs) are important intercellular signaling molecules that are essential to mammalian embryonic development. The signaling pathways between endoderm-derived gastric epithelium and the surrounding mesenchyme are largely unknown; however, the developmental expression profile of the IIIb isoform of Fgfr2 (Fgfr2b) and its main ligand, Fgf10, suggest that they may be strong candidates. Mice lacking either component (Fgfr2b-/- or Fgf10-/-) were examined to determine the role of Fgfr2b-mediated signaling during gastric organogenesis. METHODS: Stomachs from embryonic day 13.5-18.5 Fgfr2b-/-, Fgf10-/-, and wild-type littermates were collected and analyzed by conventional histology, immunohistochemistry, in situ hybridization, and electron microscopy. RESULTS: Fgfr2b-/- and Fgf10-/- fetuses had stomachs smaller than wild-type, consisting of relatively proportionate forestomach but disproportionately reduced glandular stomach, the mucosa of which has low cytoarchitectural complexity with a spiral arrangement of large mucosal folds. During mid to late fetal stages (embryonic day 15.5-18.5), epithelial differentiation to mucous and chief cell lineages was rudimentary, with no expression of several early cytodifferentiation markers including GATA4, GATA6, and H+/K+-adenosine triphosphatase and abnormal expression of members of the hedgehog family of signaling molecules. CONCLUSIONS: Fgfr2b and Fgf10 are part of a signaling network with Sonic hedgehog and Indian hedgehog that are essential to anterior-posterior and radial patterning in gastric development.

Delta-Notch signalling controls commitment to a secretory fate in the zebrafish intestine.

The transparency of the juvenile zebrafish and its genetic advantages make it an attractive model for study of cell turnover in the gut. BrdU labelling shows that the gut epithelium is renewed in essentially the same way as in mammals: the villi are lined with non-dividing differentiated cells, while cell division is confined to the intervillus pockets. New cells produced in the pockets take about 4 days to migrate out to the tips of the villi, where they die. We have generated monoclonal antibodies to identify the absorptive and secretory cells in the epithelium, and we have used these antibodies to examine the part that Delta-Notch signalling plays in producing the diversity of intestinal cell types. Several Notch receptors and ligands are expressed in the gut. In particular, the Notch ligand DeltaD (Delta1 in the mouse) is expressed in cells of the secretory lineage. In an aei mutant, where DeltaD is defective, secretory cells are overproduced. In mind bomb (mib), where all Delta-Notch signalling is believed to be blocked, almost all the cells in the 3-day gut epithelium adopt a secretory character. Thus, secretory differentiation appears to be the default in the absence of Notch activation, and lateral inhibition mediated by Delta-Notch signalling is required to generate a balanced mixture of absorptive and secretory cells. These findings demonstrate the central role of Notch signalling in the gut stem-cell system and establish the zebrafish as a model for study of the mechanisms controlling renewal of gut epithelium.

Myosin Va and microtubule-based motors are required for fast axonal retrograde transport of tetanus toxin in motor neurons.

Using a novel assay based on the sorting and transport of a fluorescent fragment of tetanus toxin, we have investigated the cytoskeletal and motor requirements of axonal retrograde transport in living mammalian motor neurons. This essential process ensures the movement of neurotrophins and organelles from the periphery to the cell body and is crucial for neuronal survival. Unlike what is observed in sympathetic neurons, fast retrograde transport in motor neurons requires not only intact microtubules, but also actin microfilaments. Here, we show that the movement of tetanus toxin-containing carriers relies on the nonredundant activities of dynein as well as kinesin family members. Quantitative kinetic analysis indicates a role for dynein as the main motor of these carriers. Moreover, this approach suggests the involvement of myosin(s) in retrograde movement. Immunofluorescence screening with isoform-specific myosin antibodies reveals colocalization of tetanus toxin-containing retrograde carriers with myosin Va. Motor neurons from homozygous myosin Va null mice showed slower retrograde transport compared with wild-type cells, establishing a unique role for myosin Va in this process. On the basis of our findings, we propose that coordination of myosin Va and microtubule-dependent motors is required for fast axonal retrograde transport in motor neurons.

The serine protease Omi/HtrA2 regulates apoptosis by binding XIAP through a reaper-like motif.

The inhibitor-of-apoptosis proteins (IAPs) play a critical role in the regulation of apoptosis by binding and inhibiting caspases. Reaper family proteins and Smac/DIABLO use a conserved amino-terminal sequence to bind to IAPs in flies and mammals, respectively, blocking their ability to inhibit caspases and thus promoting apoptosis. Here we have identified the serine protease Omi/HtrA2 as a second mammalian XIAP-binding protein with a Reaper-like motif. This protease autoprocesses to form a protein with amino-terminal homology to Smac/DIABLO and Reaper family proteins. Full-length Omi/HtrA2 is localized to mitochondria but fails to interact with XIAP. Mitochondria also contain processed Omi/HtrA2, which, following apoptotic insult, translocates to the cytosol, where it interacts with XIAP. Overexpression of Omi/HtrA2 sensitizes cells to apoptosis, and its removal by RNA interference reduces cell death. Omi/HtrA2 thus extends the set of mammalian proteins with Reaper-like function that are released from the mitochondria during apoptosis.