Patch clamp studies of single cell-fusion events mediated by a viral fusion protein

To enter cells, viruses must fuse their envelope with a host cell membrane. Fusion is mediated by specific, membrane-spanning fusion proteins, of which the influenza virus haemagglutinins (HA) are the best characterized. Several HAs have been sequenced, and the crystal structure of the major part of one HA is known. The conditions for fusion and some of the rearrangements in the HA that accompany fusion are well understood, but it remains unclear how HA causes bilayers to fuse. We have observed, in real time, unitary cell-fusion events caused by HA. Fibroblasts expressing HA were induced to fuse with red blood cells by a rapid drop in pH. Fusion was monitored by fluorescence microscopy, and by measuring the membrane conductance and capacitance of the fibroblast. The earliest event observed was the sudden opening of an aqueous pore connecting the cytoplasms of the fusing cells. Initially, the pore conductance often fluctuated between zero and approximately 600 pS, as if the pore were opening and closing repeatedly. Later, it increased over tens of seconds, as if the pore dilated. We suggest that, as in exocytosis, HA-mediated membrane fusion begins with the formation of a narrow pore. Based on the conductance, we estimate the initial diameter of the pore to be no more than twice that of a gap junction channel.     

Microorganisms seen by scanning and transmission electron microscopy in Legionnaires' disease from human lung

Summary In addition to several anomalous structures, other general forms of definitely rod-shaped microorganisms have been found by scanning and transmission electron microscopy in the lung tissue taken at autopsy from a patient who succumbed to confirmed Legionnaires' disease with extensive necrotizing lobar pneumonia. The microorganisms were greatly varied in size and shape. They were micrographed in the act of fission. These forms have been found to some extent throughout the tissue. No nickel was demonstrated, either in the lung tissue or in the microorganisms.This work has been supported in part by a Henry Ford Hospital institutional grant from the Ford Foundation and in part by the Veterans Administration Medical Center Research Funds.     

Ras regulates assembly of mitogenic signalling complexes through the effector protein IMP

The signal transduction cascade comprising Raf, mitogen-activated protein (MAP) kinase kinase (MEK) and MAP kinase is a Ras effector pathway that mediates diverse cellular responses to environmental cues and contributes to Ras-dependent oncogenic transformation. Here we report that the Ras effector protein Impedes Mitogenic signal Propagation (IMP) modulates sensitivity of the MAP kinase cascade to stimulus-dependent activation by limiting functional assembly of the core enzymatic components through the inactivation of KSR, a scaffold/adaptor protein that couples activated Raf to its substrate MEK. IMP is a Ras-responsive E3 ubiquitin ligase that, on activation of Ras, is modified by auto-polyubiquitination, which releases the inhibition of Raf-MEK complex formation. Thus, Ras activates the MAP kinase cascade through simultaneous dual effector interactions: induction of Raf kinase activity and derepression of Raf-MEK complex formation. IMP depletion results in increased stimulus-dependent MEK activation without alterations in the timing or duration of the response. These observations suggest that IMP functions as a threshold modulator, controlling sensitivity of the cascade to stimulus and providing a mechanism to allow adaptive behaviour of the cascade in chronic or complex signalling environments.     

Familial dyserythropoietic anaemia and thrombocytopenia due to an inherited mutation in GATA1

Haematopoietic development is regulated by nuclear protein complexes that coordinate lineage-specific patterns of gene expression. Targeted mutagenesis in embryonic stem cells and mice has revealed roles for the X-linked gene Gata1 in erythrocyte and megakaryocyte differentiation. GATA-1 is the founding member of a family of DNA-binding proteins that recognize the motif WGATAR through a conserved multifunctional domain consisting of two C4-type zinc fingers. Here we describe a family with X-linked dyserythropoietic anaemia and thrombocytopenia due to a substitution of methionine for valine at amino acid 205 of GATA-1. This highly conserved valine is necessary for interaction of the amino-terminal zinc finger of GATA-1 with its essential cofactor, FOG-1 (for friend of GATA-1; refs 9-12). We show that the V205M mutation abrogates the interaction between Gata-1 and Fog-1, inhibiting the ability of Gata-1 to rescue erythroid differentiation in an erythroid cell line deficient for Gata-1 (G1E). Our findings underscore the importance of FOG-1:Gata-1 associations in both megakaryocyte and erythroid development, and suggest that other X-linked anaemias or thrombocytopenias may be caused by defects in GATA1.     

Recombination signal sequences restrict chromosomal V(D)J recombination beyond the 12/23 rule

The genes encoding the variable regions of lymphocyte antigen receptors are assembled from variable (V), diversity (D) and joining (J) gene segments. V(D)J recombination is initiated by the recombinase activating gene (RAG)-1 and -2 proteins, which introduce DNA double-strand breaks between the V, D and J segments and their flanking recombination signal sequences (RSSs). Generally expressed DNA repair proteins then carry out the joining reaction. The conserved heptamer and nonamer sequences of the RSSs are separated by non-conserved spacers of 12 or 23 base pairs (forming 12-RSSs and 23-RSSs). The 12/23 rule, which is mediated at the level of RAG-1/2 recognition and cutting, specifies that V(D)J recombination occurs only between a gene segment flanked by a 12-RSS and one flanked by a 23-RSS. Vbeta segments are appended to DJbeta rearrangements, with little or no direct Vbeta to Jbeta joining, despite 12/23 compatibility of Vbeta 23-RSSs and Jbeta12-RSSs. Here we use embryonic stem cells and mice with a modified T-cell receptor (TCR)beta locus containing only one Dbeta (Dbeta1) gene segment and one Jbeta (Jbeta1) gene cluster to show that the 5' Dbeta1 12-RSS, but not the Jbeta1 12-RSSs, targets rearrangement of a diverse Vbeta repertoire. This targeting is precise and position-independent. This additional restriction on V(D)J recombination has important implications for the regulation of variable region gene assembly and repertoire development.     

IRS-2 pathways integrate female reproduction and energy homeostasis

Severe dietary restriction, catabolic states and even short-term caloric deprivation impair fertility in mammals. Likewise, obesity is associated with infertile conditions such as polycystic ovary syndrome. The reproductive status of lower organisms such as Caenorhabditis elegans is also modulated by availability of nutrients. Thus, fertility requires the integration of reproductive and metabolic signals. Here we show that deletion of insulin receptor substrate-2 (IRS-2), a component of the insulin/insulin-like growth factor-1 signalling cascade, causes female infertility. Mice lacking IRS-2 have small, anovulatory ovaries with reduced numbers of follicles. Plasma concentrations of luteinizing hormone, prolactin and sex steroids are low in these animals. Pituitaries are decreased in size and contain reduced numbers of gonadotrophs. Females lacking IRS-2 have increased food intake and obesity, despite elevated levels of leptin. Our findings indicate that insulin, together with leptin and other neuropeptides, may modulate hypothalamic control of appetite and reproductive endocrinology. Coupled with findings on the role of insulin-signalling pathways in the regulation of fertility, metabolism and longevity in C. elegans and Drosophila, we have identified an evolutionarily conserved mechanism in mammals that regulates both reproduction and energy homeostasis.