Calcium signalling in the guidance of nerve growth by netrin-1

Pathfinding by growing axons in the developing nervous system is guided by diffusible or bound factors that attract or repel the axonal growth cone. The cytoplasmic signalling mechanisms that trigger the responses of the growth cone to guidance factors are mostly unknown. Previous studies have shown that the level and temporal patterns of cytoplasmic Ca2+ can regulate the rate of growth-cone extension in vitro and in vivo. Here we report that Ca2+ also mediates the turning behaviour of the growth cones of cultured Xenopus neurons that are induced by an extracellular gradient of netrin-1, an established diffusible guidance factor in vivo. The netrin-1-induced turning response depends on Ca2+ influx through plasma membrane Ca2+ channels, as well as Ca2+-induced Ca2+ release from cytoplasmic stores. Reduction of Ca2+ signals by blocking either of these two Ca2+ sources converted the netrin-1-induced response from attraction to repulsion. Activation of Ca2+-induced Ca2+ release from internal stores with a gradient of ryanodine in the absence of netrin-1 was sufficient to trigger either attractive or repulsive responses, depending on the ryanodine concentration used. These results support the model that cytoplasmic Ca2+ signals mediate growth-cone guidance by netrin-1, and different patterns of Ca2+ elevation trigger attractive and repulsive turning responses.     

Three-dimensional structure of the ion-coupled transport protein NhaA

Ion-coupled membrane-transport proteins, or secondary transporters, comprise a diverse and abundant group of membrane proteins that are found in all organisms. These proteins facilitate solute accumulation and toxin removal against concentration gradients using energy supplied by ion gradients across membranes. NhaA is a Na+/H+ antiporter of relative molecular mass 42,000, which is found in the inner membrane of Escherichia coli, and which has been cloned and characterized. NhaA uses the H+ electrochemical gradient to expel Na+ from the cytoplasm, and functions primarily in the adaptation to high salinity at alkaline pH. Most secondary transporters, including NhaA, are predicted to have 12 transmembrane helices. Here we report the structure of NhaA, at 7 A resolution in the membrane plane and at 14 A vertical resolution, determined from two-dimensional crystals using electron cryo-microscopy. The three-dimensional map of NhaA reveals 12 tilted, bilayer-spanning helices. A roughly linear arrangement of six helices is adjacent to a compact bundle of six helices, with the density for one helix in the bundle not continuous through the membrane. The molecular organization of NhaA represents a new membrane-protein structural motif and offers the first insights into the architecture of an ion-coupled transport protein.     

Nf1;Trp53 mutant mice develop glioblastoma with evidence of strain-specific effects

Astrocytomas are the leading cause of brain cancer in humans. Because these tumours are highly infiltrative, current treatments that rely on targeting the tumour mass are often ineffective. A mouse model for astrocytoma would be a powerful tool for dissecting tumour progression and testing therapeutics. Mouse models of astrocytoma have been designed to express oncogenic proteins in astrocytes, but have had limited success due to low tumour penetrance or limited tumour progression. We present here a mouse model of astrocytomas involving mutation of two tumour-suppressor genes, Nf1 and Trp53. Humans with mutations in NF1 develop neurofibromatosis type I (NF1) and have increased risk of optic gliomas, astrocytomas and glioblastomas. The TP53 tumour suppressor is often mutated in a subset of astrocytomas that develop at a young age and progress slowly to glioblastoma (termed secondary glioblastomas, in contrast to primary glioblastomas that develop rapidly de novo). This mouse model shows a range of astrocytoma stages, from low-grade astrocytoma to glioblastoma multiforme, and may accurately model human secondary glioblastoma involving TP53 loss. This is the first reported mouse model of astrocytoma initiated by loss of tumour suppressors, rather than overexpression of transgenic oncogenes.     

Natural methyl bromide and methyl chloride emissions from coastal salt marshes

Atmospheric methyl bromide (CH3Br) and methyl chloride (CH3Cl), compounds that are involved in stratospheric ozone depletion, originate from both natural and anthropogenic sources. Current estimates of CH3Br and CH3Cl emissions from oceanic sources, terrestrial plants and fungi, biomass burning and anthropogenic inputs do not balance their losses owing to oxidation by hydroxyl radicals, oceanic degradation, and consumption in soils, suggesting that additional natural terrestrial sources may be important. Here we show that CH3Br and CH3Cl are released to the atmosphere from all vegetation zones of two coastal salt marshes. We see very large fluxes of CH3Br and CH3Cl per unit area: up to 42 and 570 micromol m(-2) d(-1), respectively. The fluxes show large diurnal, seasonal and spatial variabilities, but there is a strong correlation between the fluxes of CH3Br and those of CH3Cl, with an average molar flux ratio of roughly 1:20. If our measurements are typical of salt marshes globally, they suggest that such ecosystems, even though they constitute less than 0.1% of the global surface area, may produce roughly 10% of the total fluxes of atmospheric CH3Br and CH3Cl.     

Attention modulates synchronized neuronal firing in primate somatosensory cortex

A potentially powerful information processing strategy in the brain is to take advantage of the temporal structure of neuronal spike trains. An increase in synchrony within the neural representation of an object or location increases the efficacy of that neural representation at the next synaptic stage in the brain; thus, increasing synchrony is a candidate for the neural correlate of attentional selection. We investigated the synchronous firing of pairs of neurons in the secondary somatosensory cortex (SII) of three monkeys trained to switch attention between a visual task and a tactile discrimination task. We found that most neuron pairs in SII cortex fired synchronously and, furthermore, that the degree of synchrony was affected by the monkey's attentional state. In the monkey performing the most difficult task, 35% of neuron pairs that fired synchronously changed their degree of synchrony when the monkey switched attention between the tactile and visual tasks. Synchrony increased in 80% and decreased in 20% of neuron pairs affected by attention.     

A clonogenic common myeloid progenitor that gives rise to all myeloid lineages

Haematopoietic stem cells give rise to progeny that progressively lose self-renewal capacity and become restricted to one lineage. The points at which haematopoietic stem cell-derived progenitors commit to each of the various lineages remain mostly unknown. We have identified a clonogenic common lymphoid progenitor that can differentiate into T, B and natural killer cells but not myeloid cells. Here we report the prospective identification, purification and characterization, using cell-surface markers and flow cytometry, of a complementary clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Common myeloid progenitors give rise to either megakaryocyte/erythrocyte or granulocyte/macrophage progenitors. Purified progenitors were used to provide a first-pass expression profile of various haematopoiesis-related genes. We propose that the common lymphoid progenitor and common myeloid progenitor populations reflect the earliest branch points between the lymphoid and myeloid lineages, and that the commitment of common myeloid progenitors to either the megakaryocyte/erythrocyte or the granulocyte/macrophage lineages are mutually exclusive events.     

The S receptor kinase determines self-incompatibility in Brassica stigma

The self-incompatibility possessed by Brassica is an intraspecific reproductive barrier by which the stigma rejects self-pollen but accepts non-self-pollen for fertilization. The molecular/biochemical bases of recognition and rejection have been intensively studied. Self-incompatibility in Brassica is sporophytically controlled by the polymorphic S locus. Two tightly linked polymorphic genes at the S locus, S receptor kinase gene (SRK) and S locus glycoprotein gene (SLG), are specifically expressed in the papillar cells of the stigma, and analyses of self-compatible lines of Brassica have suggested that together they control stigma function in self-incompatibility interactions. Here we show, by transforming self-incompatible plants of Brassica rapa with an SRK28 and an SLG28 transgene separately, that expression of SRK28 alone, but not SLG28 alone, conferred the ability to reject self (S28)-pollen on the transgenic plants. We also show that the ability of SRK28 to reject S28 pollen was enhanced by SLG28. We conclude that SRK alone determines S haplotype specificity of the stigma, and that SLG acts to promote a full manifestation of the self-incompatibility response.     

Structure of a ligand-binding intermediate in wild-type carbonmonoxy myoglobin

Small molecules such as NO, O2, CO or H2 are important biological ligands that bind to metalloproteins to function crucially in processes such as signal transduction, respiration and catalysis. A key issue for understanding the regulation of reaction mechanisms in these systems is whether ligands gain access to the binding sites through specific channels and docking sites, or by random diffusion through the protein matrix. A model system for studying this issue is myoglobin, a simple haem protein. Myoglobin has been studied extensively by spectroscopy, crystallography, computation and theory. It serves as an aid to oxygen diffusion but also binds carbon monoxide, a byproduct of endogenous haem catabolism. Molecular dynamics simulations, random mutagenesis and flash photolysis studies indicate that ligand migration occurs through a limited number of pathways involving docking sites. Here we report the 1.4 A resolution crystal structure of a ligand-binding intermediate in carbonmonoxy myoglobin that may have far-reaching implications for understanding the dynamics of ligand binding and catalysis.