mRNA binding protein mrnp 41 localizes to both nucleus and cytoplasm

We have identified and molecularly characterized a human protein with a Mr of 40,880 Da. After UV irradiation of HeLa cells, this protein was cross-linked to poly(A)-containing mRNA and was therefore designated mrnp 41 (for mRNA binding protein of 41 kDa). Cell fractionation and immunoblotting showed mrnp 41 in both the cytoplasm and the nucleus and particularly in the nuclear envelope. Immunofluorescence microscopy localized mrnp 41 to distinct foci in the nucleoplasm, to the nuclear rim, and to meshwork-like structures throughout the cytoplasm. The cytoplasmic meshwork staining was disrupted by prior treatment of cells with the actin filament- or microtubule-disrupting drugs cytochalasin or nocodazole, respectively, suggesting association of mrnp 41 with the cytoskeleton. Double immunofluorescence with antibodies against mrnp 41 and the cytoplasmic poly(A) binding protein showed colocalization to the cytoplasmic meshwork. Immunogold electronmicroscopy confirmed mrnp 41’s cytoplasmic and nucleoplasmic localization and revealed a striking labeling of nuclear pore complexes. Together these data suggest that mrnp 41 may function in nuclear export of mRNPs and/or in cytoplasmic transport on, or attachment to, the cytoskeleton. Consistent with a role of mrnp 41 in nuclear export are previous reports that mutations in homologs of mrnp 41 in Schizosaccharomyces pombe, designated Rae1p, or in Saccharomyces cerevisiae, designated Gle2p, result in mRNA accumulation in the nucleus although it is presently not known whether these homologs are mRNA binding proteins as well.

Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling

We have established a differential peptide display method, based on a mass spectrometric technique, to detect peptides that show semiquantitative changes in the neurointermediate lobe (NIL) of individual rats subjected to salt-loading. We employed matrix-assisted laser desorption/ionization mass spectrometry, using a single-reference peptide in combination with careful scanning of the whole crystal rim of the matrix-analyte preparation, to detect in a semiquantitative manner the molecular ions present in the unfractionated NIL homogenate. Comparison of the mass spectra generated from NIL homogenates of salt-loaded and control rats revealed a selective and significant decrease in the intensities of several molecular ion species of the NIL homogenates from salt-loaded rats. These ion species, which have masses that correspond to the masses of oxytocin, vasopressin, neurophysins, and an unidentified putative peptide, were subsequently chemically characterized. We confirmed that the decreased molecular ion species are peptides derived exclusively from propressophysin and prooxyphysin (i.e., oxytocin, vasopressin, and various neurophysins). The putative peptide is carboxyl-terminal glycopeptide. The carbohydrate moiety of the latter peptide was determined by electrospray tandem MS as bisected biantennary Hex3HexNAc5Fuc. This posttranslational modification accounts for the mass difference between the predicted mass of the peptide based on cDNA studies and the measured mass of the mature peptide.

Karyopherin β2 mediates nuclear import of a mRNA binding protein

We have cloned and sequenced cDNA for human karyopherin β2, also known as transportin. In a solution binding assay, recombinant β2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1’s previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin β1, karyopherin β2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both β1 and β2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, β2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of β2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of β2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin β2 inhibited karyopherin α/β1-mediated import of a classical NLS containing substrate and, vice versa, β1 inhibited β2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of β1 and β2 to repeat nucleoporins.

An equilibrium statistical model for the spreading phase of open-ocean convection

A “most probable state” equilibrium statistical theory for random distributions of hetons in a closed basin is developed here in the context of two-layer quasigeostrophic models for the spreading phase of open-ocean convection. The theory depends only on bulk conserved quantities such as energy, circulation, and the range of values of potential vorticity in each layer. The simplest theory is formulated for a uniform cooling event over the entire basin that triggers a homogeneous random distribution of convective towers. For a small Rossby deformation radius typical for open-ocean convection sites, the most probable states that arise from this theory strongly resemble the saturated baroclinic states of the spreading phase of convection, with a stabilizing barotropic rim current and localized temperature anomaly.

Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission

The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90–110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details.

The cranium of Parapithecus grangeri, an Egyptian Oligocene anthropoidean primate

A nearly complete skull of Parapithecus grangeri from the early Oligocene of Egypt is described. The specimen is relatively undistorted and is undoubtedly the most complete higher primate skull yet found in the African Oligocene, which also makes it the most complete Oligocene primate cranium worldwide. Belonging in superfamily Parapithecoidea, a group regarded by some as the sister group to all other Anthropoidea, this skull reveals important information about the radiation of stem anthropoideans. This cranium is about 15% larger than size estimates based on a fragmentary cranium of its contemporary and close relative Apidium phiomense. It is about the same size as that of the gray gentle lemur, Hapalemur griseus, or of platyrrhines such as the owl monkey, Aotus trivirgatus, or the titi monkey, Callicebus torquatus. Comparatively small orbits and size differences in jaws and teeth show it was both diurnal and dimorphic. This is the only specimen of the species that shows (from sockets) that there were four small upper incisors. Several mandibular specimens of the species establish that there were no permanent lower incisors and that the symphysis was fused. Like other early anthropoideans this species possessed a lower encephalization quotient and less-developed orbital frontality than later anthropoideans. There is full postorbital closure and fusion of the metopic suture, and the ectotympanic forms a rim to the auditory aperture. A probable frontal/alisphenoid contact is a potentially derived resemblance to Catarrhini. A proposed separate genus for the species P. grangeri is not sustained.

Multiple vesiculoviral matrix proteins inhibit both nuclear export and import

The matrix (M) protein of vesicular stomatitis virus inhibits both nuclear import and export. Here, we demonstrate that this inhibitory property is conserved between the M proteins from two other vesiculoviruses, chandipura virus and spring viremia carp virus. All three M proteins completely block nuclear transport of spliced mRNA, small nuclear RNAs, and small nuclear ribonucleoproteins and slow the nuclear transport of many other cargoes. In all cases where transport was merely slowed by the M proteins, the chandipura virus M protein had the strongest inhibitory activity. When expressed in transfected HeLa cells, active M proteins displayed prominent association with the nuclear rim. Moreover, mutation of a conserved methionine abolished both the inhibitory activity and efficient targeting of the M proteins to the nuclear rim. We propose that all of the vesiculoviral M proteins associate with the same nuclear target, which is likely to be a component of the nuclear pore complex.

Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K+ channel KAT1.

KAT1 is a voltage-dependent inward rectifying K+ channel cloned from the higher plant Arabidopsis thaliana [Anderson, J. A., Huprikar, S. S., Kochian, L. V., Lucas, W. J. & Gaber, R. F. (1992) Proc. Natl. Acad. Sci. USA 89, 3736-3740]. It is related to the Shaker superfamily of K+ channels characterized by six transmembrane spanning domains (S1-S6) and a putative pore-forming region between S5 and S6 (H5). The 115 region between Pro-247 and Pro-271 in KAT1 contains 14 additional amino acids when compared with Shaker [Aldrich, R. W. (1993) Nature (London) 362, 107-108]. We studied various point mutations introduced into H5 to determine whether voltage-dependent plant and animal K+ channels share similar pore structures. Through heterologous expression in Xenopus oocytes and voltage-clamp analysis combined with phenotypic analysis involving a potassium transport-defective Saccharomyces cerevisiae strain, we investigated the selectivity filter of the mutants and their susceptibility toward inhibition by cesium and calcium ions. With respect to electrophysiological properties, KAT1 mutants segregated into three groups: (i) wild-type-like channels, (ii) channels modified in selectivity and Cs+ or Ca2+ sensitivity, and (iii) a group that was additionally affected in its voltage dependence. Despite the additional 14 amino acids in H5, this motif in KAT1 is also involved in the formation of the ion-conducting pore because amino acid substitutions at Leu-251, Thr-256, Thr-259, and Thr-260 resulted in functional channels with modified ionic selectivity and inhibition. Creation of Ca2+ sensitivity and an increased susceptibility to Cs+ block through mutations within the narrow pore might indicate that both blockers move deeply into the channel. Furthermore, mutations close to the rim of the pore affecting the half-activation potential (U1/2) indicate that amino acids within the pore either interact with the voltage sensor or ion permeation feeds back on gating.

Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice.

Synapsin I has been proposed to be involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis. To further understand the role of synapsin I in the function of adult nerve terminals, we studied synapsin I-deficient mice generated by homologous recombination. The organization of synaptic vesicles at presynaptic terminals of synapsin I-deficient mice was markedly altered: densely packed vesicles were only present in a narrow rim at active zones, whereas the majority of vesicles were dispersed throughout the terminal area. This was in contrast to the organized vesicle clusters present in terminals of wild-type animals. Release of glutamate from nerve endings, induced by K+,4-aminopyridine, or a Ca2+ ionophore, was markedly decreased in synapsin I mutant mice. The recovery of synaptic transmission after depletion of neurotransmitter by high-frequency stimulation was greatly delayed. Finally, synapsin I-deficient mice exhibited a strikingly increased response to electrical stimulation, as measured by electrographic and behavioral seizures. These results provide strong support for the hypothesis that synapsin I plays a key role in the regulation of nerve terminal function in mature synapses.

Protein export from the nucleus requires the GTPase Ran and GTP hydrolysis.

Nuclei of digitonin-permeabilized cells that had been preloaded with a model transport substrate in a cytosol-dependent import reaction were subsequently incubated to investigate which conditions would result in export of transport substrate. We found that up to 80% of the imported substrate was exported when recombinant human Ran and GTP were present in the export reaction. Ran-mediated export was inhibited by nonhydrolyzable GTP analogs and also by wheat germ agglutinin but was unaffected by a nonhydrolyzable ATP analog. Moreover, a recombinant human Ran mutant that was deficient in its GTPase activity inhibited export. These data indicate that export of proteins from the nucleus requires Ran and GTP hydrolysis but not ATP hydrolysis. We also found that digitonin-permeabilized cells were depleted of their endogenous nuclear Ran, thus allowing detection of Ran as a limiting factor for export. In contrast, most endogenous karyopherin alpha was retained in nuclei of digitonin-permeabilized cells. Unexpectedly, exogenously added, fluorescently labeled Ran, although it accessed the nuclear interior, was found to dock at the nuclear rim in a punctate pattern, suggesting the existence of Ran-binding sites at the nuclear pore complex.