ApoNifH functions in iron–molybdenum cofactor synthesis and apodinitrogenase maturation

NifH (dinitrogenase reductase) has three important roles in the nitrogenase enzyme system. In addition to its role as the obligate electron donor to dinitrogenase, NifH is required for the iron–molybdenum cofactor (FeMo-co) synthesis and apodinitrogenase maturation. We have investigated the requirement of the Fe–S cluster of NifH for these processes by preparing apoNifH. The 4Fe–4S cluster of NifH was removed by chelation of the cluster with α, α′-bipyridyl. The resulting apoNifH was tested in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions and was found to function in both these processes. Thus, the presence of a redox active 4Fe–4S cluster in NifH is not required for its function in FeMo-co synthesis and in apodinitrogenase maturation. This, in turn, implies that the role of NifH in these processes is not one of electron transfer or of iron or sulfur donation.

Xenopus Cdc6 confers sperm binding competence to oocytes without inducing their maturation

Amphibian eggs normally require meiotic maturation to be competent for fertilization. A necessary prerequisite for this event is sperm binding, and we show that under normal physiological conditions this property is acquired at, but not before, meiotic maturation. Immature oocytes do not bind sperm, but injection of total egg poly(A)+ mRNA into immature oocytes confers sperm binding in the absence of meiotic maturation. Using an expression cloning approach we have isolated a single cDNA from egg poly(A)+ mRNA that can induce sperm binding in immature oocytes. The cDNA was found to encode Xenopus Cdc6, a protein that previously has been shown to function in initiation of DNA replication and cell cycle control. This unanticipated finding provides evidence of a link between a regulator of the cell cycle and alterations in cell surface properties that affect gamete binding.

Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation

Changes in DNA methylation during tobacco pollen development have been studied by confocal fluorescence microscopy using a monoclonal anti-5-methylcytosine (anti-m5C) antibody and a polyclonal anti-histone H1 (anti-histone) antibody as an internal standard. The specificity of the anti-m5C antibody was demonstrated by a titration series against both single-stranded DNA and double-stranded DNA substrates in either the methylated or unmethylated forms. The antibody was found to show similar kinetics against both double- and single-stranded DNA, and the fluorescence was proportional to the amount of DNA used. No signal was observed with unmethylated substrates. The extent of methylation of the two pollen nuclei remained approximately constant after the mitotic division that gave rise to the vegetative and generative nuclei. However, during the subsequent development of the pollen, the staining of the generative nucleus decreased until it reached a normalized value of \documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}\frac{1}{5}\end{equation*}\end{document} of that of the vegetative nucleus. The use of a confocal microscope makes these data independent of possible focusing artefacts. The anti-histone antibody was used as a control to show that, while the antibody staining directed against 5-methylcytosine changed dramatically during pollen maturation, the histone signal did not. We observed the existence of structural dimorphism amongst tobacco pollen grains, the majority having three pollen apertures and the rest with four. However, the methylation changes observed occurred to the same extent in both subclasses.

Nuclear hourglass technique: An approach that detects electrically open nuclear pores in Xenopus laevis oocyte

Nuclear pore complexes (NPCs) mediate both active transport and passive diffusion across the nuclear envelope (NE). Determination of NE electrical conductance, however, has been confounded by the lack of an appropriate technical approach. The nuclear patch clamp technique is restricted to preparations with electrically closed NPCs, and microelectrode techniques fail to resolve the extremely low input resistance of large oocyte nuclei. To address the problem, we have developed an approach for measuring the NE electrical conductance of Xenopus laevis oocyte nuclei. The method uses a tapered glass tube, which narrows in its middle part to 2/3 of the diameter of the nucleus. The isolated nucleus is sucked into the narrow part of the capillary by gentle fluid movement, while the resulting change in electrical resistance is monitored. NE electrical conductance was unexpectedly large (7.9 ± 0.34 S/cm2). Evaluation of NPC density by atomic force microscopy showed that this conductance corresponded to 3.7 × 106 NPCs. In contrast to earlier conclusions drawn from nuclear patch clamp experiments, NPCs were in an electrically “open” state with a mean single NPC electrical conductance of 1.7 ± 0.07 nS. Enabling or blocking of active NPC transport (accomplished by the addition of cytosolic extracts or gp62-directed antibodies) revealed this large NPC conductance to be independent of the activation state of the transport machinery located in the center of NPCs. We conclude that peripheral channels, which are presumed to reside in the NPC subunits, establish a high ionic permeability that is virtually independent of the active protein transport mechanism.

Molecular dynamics and free-energy calculations applied to affinity maturation in antibody 48G7

We investigated the relative free energies of hapten binding to the germ line and mature forms of the 48G7 antibody Fab fragments by applying a continuum model to structures sampled from molecular dynamics simulations in explicit solvent. Reasonable absolute and very good relative free energies were obtained. As a result of nine somatic mutations that do not contact the hapten, the affinity-matured antibody binds the hapten >104 tighter than the germ line antibody. Energetic analysis reveals that van der Waals interactions and nonpolar contributions to solvation are similar and drive the formations of both the germ line and mature antibody–hapten complexes. Affinity maturation of the 48G7 antibody therefore appears to occur through reorganization of the combining site geometry in a manner that optimizes the balance of gaining favorable electrostatic interactions with the hapten and losing those with solvent during the binding process. As reflected by lower rms fluctuations in the antibody–hapten complex, the mature complex undergoes more restricted fluctuations than the germ line complex. The dramatically increased affinity of the 48G7 antibody over its germ line precursor is thus made possible by electrostatic optimization.

Identification of an early T cell progenitor for a pathway of T cell maturation in the bone marrow

We have identified a rare (≈0.05–0.1%) population of cells (Thy-1hiCD16+CD44hiCD2−TCRαβ−B220−Mac-1−NK1.1−) in the adult mouse bone marrow that generates CD4+ and CD8+ TCRαβ+ T cells after tissue culture for 48 hr in the presence of Ly5 congenic marrow cells. The essential stages in the maturation of the progenitors were determined; the stages included an early transition from CD2−CD16+CD44hiTCRαβ− to CD2+CD16int/−CD44int/−TCRαβ− cells, and a later transition to CD4+CD8+TCRαβ+ double-positive T cells that rapidly generate the CD4+ and CD8+ single-positive T cells. The maturation of the progenitors is almost completely arrested at the CD2+TCRαβ− stage by the presence of mature T cells at the initiation of cultures. This alternate pathway is supported by the marrow microenvironment; it recapitulates critical intermediary steps in intrathymic T cell maturation.

Induction of maturation of human blood dendritic cell precursors by measles virus is associated with immunosuppression

As well as inducing a protective immune response against reinfection, acute measles is associated with a marked suppression of immune functions against superinfecting agents and recall antigens, and this association is the major cause of the current high morbidity and mortality rate associated with measles virus (MV) infections. Dendritic cells (DCs) are antigen-presenting cells crucially involved in the initiation of primary and secondary immune responses, so we set out to define the interaction of MV with these cells. We found that both mature and precursor human DCs generated from peripheral blood monocytic cells express the major MV protein receptor CD46 and are highly susceptible to infection with both MV vaccine (ED) and wild-type (WTF) strains, albeit with different kinetics. Except for the down-regulation of CD46, the expression pattern of functionally important surface antigens on mature DCs was not markedly altered after MV infection. However, precursor DCs up-regulated HLA-DR, CD83, and CD86 within 24 h of WTF infection and 72 h after ED infection, indicating their functional maturation. In addition, interleukin 12 synthesis was markedly enhanced after both ED and WTF infection in DCs. On the other hand, MV-infected DCs strongly interfered with mitogen-dependent proliferation of freshly isolated peripheral blood lymphocytes in vitro. These data indicate that the differentiation of effector functions of DCs is not impaired but rather is stimulated by MV infection. Yet, mature, activated DCs expressing MV surface antigens do give a negative signal to inhibit lymphocyte proliferation and thus contribute to MV-induced immunosuppression.

Abnormal dendritic spines in fragile X knockout mice: Maturation and pruning deficits

Fragile X syndrome arises from blocked expression of the fragile X mental retardation protein (FMRP). Golgi-impregnated mature cerebral cortex from fragile X patients exhibits long, thin, tortuous postsynaptic spines resembling spines observed during normal early neocortical development. Here we describe dendritic spines in Golgi-impregnated cerebral cortex of transgenic fragile X gene (Fmr1) knockout mice that lack expression of the protein. Dendritic spines on apical dendrites of layer V pyramidal cells in occipital cortex of fragile X knockout mice were longer than those in wild-type mice and were often thin and tortuous, paralleling the human syndrome and suggesting that FMRP expression is required for normal spine morphological development. Moreover, spine density along the apical dendrite was greater in the knockout mice, which may reflect impaired developmental organizational processes of synapse stabilization and elimination or pruning.

Regulation of Cadherin Function by Rho and Rac: Modulation by Junction Maturation and Cellular Context

Cadherins are cell–cell adhesion receptors whose adhesive function requires their association with the actin cytoskeleton via proteins called catenins. The small guanosine triphosphatases (GTPases), Rho and Rac, are intracellular proteins that regulate the formation of distinct actin structures in different cell types. In keratinocytes and in other epithelial cells, Rho and Rac activities are required for E-cadherin function. Here we show that the regulation of cadherin adhesiveness by the small GTPases is influenced by the maturation status of the junction and the cellular context. E-cadherin localization was disrupted in mature keratinocyte junctions after inhibition of Rho and Rac. However, an incubation of 2 h was required after GTPase inhibition, when compared with newly established E-cadherin contacts (30 min). Regarding other cadherin receptors, P-cadherin was effectively removed from mature keratinocytes junctions by blocking Rho or Rac. In contrast, VE-cadherin localization at endothelial junctions was independent of Rho/Rac activity. We demontrate that the insensitivity of VE-cadherin to inhibition of Rho and Rac was not due to the maturation status of endothelial junction, but rather the cellular background: when transfected into CHO cells, the localization of VE-cadherin was perturbed by inhibition of Rho proteins. Our results suggest that the same stimuli may have different activity in regulating the paracellular activity in endothelial and epithelial cells. In addition, we uncovered possible roles for the small GTPases during the establishment of E-cadherin–dependent contacts. In keratinocytes, Rac activation by itself cannot promote accumulation of actin at the cell periphery in the absence of cadherin-dependent contacts. Moreover, neither Rho nor Rac activation was sufficient to redistribute cadherin molecules to cell borders, indicating that redistribution results mostly from the homophilic binding of the receptors. Our results point out the complexity of the regulation of cadherin-mediated adhesion by the small GTPases, Rho and Rac.

Genetic Dissection of Endocytic Trafficking in Drosophila Using a Horseradish Peroxidase-Bride of Sevenless Chimera: hook Is Required for Normal Maturation of Multivesicular Endosomes

Mutations in the hook gene alter intracellular trafficking of internalized ligands in Drosophila. To dissect this defect in more detail, we developed a new approach to visualize the pathway taken by the Bride of Sevenless (Boss) ligand after its internalization into R7 cells. A chimeric protein consisting of HRP fused to Boss (HRP-Boss) was expressed in R8 cells. This chimera was fully functional: it rescued the boss mutant phenotype, and its trafficking was indistinguishable from that of the wild-type Boss protein. The HRP activity of the chimera was used to follow HRP-Boss trafficking on the ultrastructural level through early and late endosomes in R7 cells. In both wild-type and hook mutant eye disks, HRP-Boss was internalized into R7 cells. In wild-type tissue, Boss accumulated in mature multivesicular bodies (MVBs) within R7 cells; such accumulation was not observed in hook eye disks, however. Quantitative electron microscopy revealed a loss of mature MVBs in hook mutant tissue compared with wild type, whereas more than twice as many multilammelar late endosomes were detected. Our genetic analysis indicates that Hook is required late in endocytic trafficking to negatively regulate delivery from mature MVBs to multilammelar late endosomes and lysosomes.

Receptor-mediated Endocytosis in the Caenorhabditis elegans Oocyte

The Caenorhabditis elegans oocyte is a highly amenable system for forward and reverse genetic analysis of receptor-mediated endocytosis. We describe the use of transgenic strains expressing a vitellogenin::green fluorescent protein (YP170::GFP) fusion to monitor yolk endocytosis by the C. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. elegans predicted proteins homologous to vertebrate endocytosis factors using RNA-mediated interference. We show that the basic components and pathways of endocytic trafficking are conserved between C. elegans and vertebrates, and that this system can be used to test the endocytic functions of any new gene. We also used the YP170::GFP assay to identify rme (receptor-mediated endocytosis) mutants. We describe a new member of the low-density lipoprotein receptor superfamily, RME-2, identified in our screens for endocytosis defective mutants. We show that RME-2 is the C. elegans yolk receptor.

Estrogen receptor specificity in the regulation of skeletal growth and maturation in male mice

Androgens may regulate the male skeleton directly through a stimulation of androgen receptors or indirectly through aromatization of androgens into estrogen and, thereafter, through stimulation of estrogen receptors (ERs). The relative importance of ER subtypes in the regulation of the male skeleton was studied in ERα-knockout (ERKO), ERβ-knockout (BERKO), and double ERα/β-knockout (DERKO) mice. ERKO and DERKO, but not BERKO, demonstrated decreased longitudinal as well as radial skeletal growth associated with decreased serum levels of insulin-like growth factor I. Therefore, ERα, but not ERβ, mediates important effects of estrogen in the skeleton of male mice during growth and maturation.

Complex formation between stage-specific oocyte factors and a Xenopus mRNA localization element

It is a long-standing proposal that localization of maternal factors in eggs can provide the basis for pattern formation in the early embryo. The localized information can be stored as RNA, one example being Vg1 RNA, which is localized exclusively to the vegetal hemisphere of Xenopus oocytes and eggs. Localization of Vg1 mRNA is directed by a 340-nt sequence element contained within its 3′ untranslated region. To understand the mechanism of localization, I have tested whether factors from the oocyte interact specifically with the RNA localization sequence. Results presented here show that a set of oocyte proteins form complexes with the localization element both in vitro and in vivo. These proteins are specifically enriched in the stages of oogenesis during which localization occurs and recognize sub-elements of the RNA localization element that are essential for localization in vivo. These data suggest that formation of a localization-specific RNA–protein complex may be the first step in directing Vg1 mRNA to its subcellular destination.

Length suppression in histone messenger RNA 3′-end maturation: Processing defects of insertion mutant premessenger RNAs can be compensated by insertions into the U7 small nuclear RNA

Efficient 3′-end processing of cell cycle-regulated mammalian histone premessenger RNAs (pre-mRNAs) requires an upstream stem–loop and a histone downstream element (HDE) that base pairs with the U7 small ribonuclearprotein. Insertions between these elements have two effects: the site of cleavage moves in concert with the HDE and processing efficiency declines. We used Xenopus oocytes to ask whether compensatory length insertions in the human U7 RNA could restore the fidelity and efficiency of processing of mouse histone insertion pre-mRNAs. An insertion of 5 nt into U7 RNA that extends its complementary to the HDE compensated for both defects in processing of a 5-nt insertion substrate; a noncomplementary insertion into U7 did not. Yet, the noncomplementary insertion mutant U7 was shown to be active on insertion substrates further mutated to allow base pairing. Our results suggest that the histone pre-mRNA becomes rigidified upstream of its HDE, allowing the bound U7 small ribonucleoprotein to measure from the HDE to the cleavage site. Such a mechanism may be common to other RNA measuring systems. To our knowledge, this is the first demonstration of length suppression in an RNA processing system.