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Recent studies in Xenopus egg extracts indicate that the small G protein Ran has a central role in spindle assembly and nuclear envelope reformation. We determined Ran localization and dynamics in cells during M phase. By immunofluorescence, Ran is accumulated on the chromosomes of meiosis-II-arrested Xenopus eggs. In living cells, fluorescently labeled Ran associated with the chromosomes in Xenopus and remained associated during anaphase when eggs were artificially activated. Fluorescent Ran associated with chromosomes in mouse eggs, during meiotic maturation and early embryonic divisions in starfish, and to a lesser degree during mitosis of a cultured mammalian cell line. Chromosomal Ran undergoes constant flux. From photobleach experiments in immature starfish oocytes, chromosomal Ran has a k(off) of approximately 0.06 second(-1), and binding analysis suggests that there is a single major site. The chromosomal interactions may serve to keep Ran-GTP in the vicinity of the chromosomes for spindle assembly and nuclear envelope reformation.

We have identified a novel mammalian protein, MIR1, with microtubule-binding activity. MIR1 is a relative of MID1/midin, the protein implicated in Opitz G/BBB syndrome. In tissue culture cells, MIR1 is enriched at the centrosome. MIR1 dissociates from centrosomes at the G2/M transition and is recruited back to spindle poles during anaphase. When overexpressed during interphase, MIR1 binds along microtubule filaments, which become stabilized, bundled and detached from the centrosome. In mitosis, overexpressed MIR1 dissociates from microtubules but still affects the normally focused localization of gamma-tubulin in spindle poles. Tight binding to microtubules in interphase appears to require an oligomeric state of MIR1, and phosphorylation in mitosis at predicted cyclin-dependent kinase (cdk) sites weakens the interaction.

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.

The nuclear envelope (NE) is a distinct subdomain of the ER, but few membrane components have been described that are specific to it. We performed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER. We confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subcompartments of mammalian cells, including the NE. With this approach, we identified a new NE membrane protein, named nurim. Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus. Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins. Thus, nurim is a new type of NE membrane protein that is localized to the NE by a distinct mechanism.

We have examined the role of FGF signalling in the development of muscle and notochord and in the expression of early mesodermal markers in Xenopus embryos. Disruption of the FGF signalling pathway by expression of a dominant negative construct of the FGF receptor (XFD) generally results in gastrulation defects that are later evident in the formation of the trunk and tail, though head structures are formed nearly normally. These defects are reflected in the loss of notochord and muscle. Even in embryos that show mild defects and gastrulate properly, muscle formation is impaired, suggesting that morphogenesis and tissue differentiation each depend on FGF. The XFD protein inhibits the expression of the immediate early gene brachyury throughout the marginal zone, including the dorsal side; it does not, however, inhibit the dorsal lip marker goosecoid, which is expressed in the first involuting mesoderm at the dorsal side that will underlie the head. The XFD protein also inhibits Xpo expression, an immediate early marker of ventral and lateral mesoderm. These results suggest that FGF is involved in the earliest events of most mesoderm induction that occur before gastrulation and that the early dorsal mesoderm is already composed of two cell populations that differ in their requirements for FGF.

From nucleotide sequencing analyses, the A. caulinodans nifA gene seems to be under dual control by the Ntr (in response to available N) and Fnr (in response to available O2) transcriptional activation/repression systems. Because it fixes N2 in two contexts, the Ntr system might regulate A. caulinodans nif gene expression ex planta, while the Fnr system might similarly regulate in planta. As nifA upstream-activating elements, we have identified: (i) a gpNifA binding site allowing autogenous nifA regulation, (ii) an Ntr-dependent transcription start, presumably the target of gpNifA activation, and (iii) an "anaerobox" tetradecameric nucleotide sequence that is precisely conserved among O2 regulated enteric bacterial genes controlled by the gpFnr transcriptional activator. Because it is precisely positioned upstream of enteric bacterial transcriptional starts, the "anaerobox" sequence may constitute the gpFnr DNA binding site. If so, then a second, Ntr-independent nifA transcription start may exist. We have also deduced the A. caulinodans nifA open reading frame and have compared the gene product (gpNifA) with those of other N2-fixing organisms. These proteins exhibit strongly conserved motifs: (i) sites conserved among ATP-binding proteins, (ii) an interdomain linker region, and (iii) a C-terminal alpha-helix-turn-alpha-helix DNA binding site.