Fibulin-5 is an elastin-binding protein essential for elastic fibre development in vivo.

Extracellular elastic fibres provide mechanical elasticity to tissues and contribute towards the processes of organ remodelling by affecting cell-cell signalling. The formation of elastic fibres requires the assembly and crosslinking of tropoelastin monomers, and organization of the resulting insoluble elastin matrix into functional fibres. The molecules and mechanisms involved in this process are unknown. Fibulin-5 (also known as EVEC/DANCE) is an extracellular matrix protein abundantly expressed in great vessels and cardiac valves during embryogenesis, and in many adult tissues including the aorta, lung, uterus and skin, all of which contain abundant elastic fibres. Here we show that fibulin-5 is a calcium-dependent, elastin-binding protein that localizes to the surface of elastic fibres in vivo. fibulin-5-/- mice develop marked elastinopathy owing to the disorganization of elastic fibres, with resulting loose skin, vascular abnormalities and emphysematous lung. This phenotype, which resembles the cutis laxa syndrome in humans, reveals a critical function for fibulin-5 as a scaffold protein that organizes and links elastic fibres to cells. This function may be mediated by the RGD motif in fibulin-5, which binds to cell surface integrins, and the Ca2+-binding epidermal growth factor (EGF) repeats, which bind elastin.     

Rab5 is necessary for the biogenesis of the endolysosomal system in vivo

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.     

T-box gene tbx5 is essential for formation of the pectoral limb bud

The T-box genes Tbx4 and Tbx5 have been shown to have key functions in the specification of the identity of the vertebrate forelimb (Tbx5) and hindlimb (Tbx4). Here we show that in zebrafish, Tbx5 has an additional early function that precedes the formation of the limb bud itself. Functional knockdown of zebrafish tbx5 through the use of an antisense oligonucleotide resulted in a failure to initiate fin bud formation, leading to the complete loss of pectoral fins. The function of the tbx5 gene in the development of zebrafish forelimbs seems to involve the directed migration of individual lateral-plate mesodermal cells into the future limb-bud-producing region. The primary defect seen in the tbx5-knockdown phenotype is similar to the primary defects described in known T-box-gene mutants such as the spadetail mutant of zebrafish and the Brachyury mutant of the mouse, which both similarly exhibit an altered migration of mesodermal cells. A common function for many of the T-box genes might therefore be in mediating the proper migration and/or changes in adhesive properties of early embryonic cells.     

Bifunctional enzyme FBPase/SBPase is essential for photoautotrophic growth in cyanobacterium Synechocystis sp. PCC 6803

From a random insertion mutant library of Synechocystis sp. PCC 6803, a mutant defective in photoautotrophic growth was obtained. The interrupted gene was identified to be slr2094 （fbpl）, which encodes the fructose-1,6-biphosphatase （FBPase）/sedoheptulose-1,7-biphosphatase （SBPase） bifunctional enzyme （F-I）. Two other independently constructed slr2094 mutants showed an identical phenotype. The FBPase activity was found to be virtually lacking in an slr2094 mutant, which was sensitive to light under mixotrophic growth conditions. These results indicate that slr2094 is the only active FBPase-encoding gene in this cyanobacterium. Inactivation of photosystem II by interrupting psbB in slr2094 mutant alleviated the sensitiveness to light. This report provides the direct genetic evidence for the essential role of F-I in the photosynthesis of Synechocystis sp. PCC 6803.     

Repressor activity of Headless/Tcf3 is essential for vertebrate head formation

The vertebrate organizer can induce a complete body axis when transplanted to the ventral side of a host embryo by virtue of its distinct head and trunk inducing properties. Wingless/Wnt antagonists secreted by the organizer have been identified as head inducers. Their ectopic expression can promote head formation, whereas ectopic activation of Wnt signalling during early gastrulation blocks head formation. These observations suggest that the ability of head inducers to inhibit Wnt signalling during formation of anterior structures is what distinguishes them from trunk inducers that permit the operation of posteriorizing Wnt signals. Here we describe the zebrafish headless (hdl) mutant and show that its severe head defects are due to a mutation in T-cell factor-3 (Tcf3), a member of the Tcf/Lef family. Loss of Tcf3 function in the hdl mutant reveals that hdl represses Wnt target genes. We provide genetic evidence that a component of the Wnt signalling pathway is essential in vertebrate head formation and patterning.     

ICOS is essential for effective T-helper-cell responses

The outcome of T-cell responses after T-cell encounter with specific antigens is modulated by co-stimulatory signals, which are required for both lymphocyte activation and development of adaptive immunity. ICOS, an inducible co-stimulator with homology to CD28, is expressed on activated, but not resting T cells, and shows T-cell co-stimulatory function in vitro. ICOS binds specifically to its counter-receptor B7RP-1 (refs 5,6,7), but not to B7-1 or B7-2. Here we provide in vivo genetic evidence that ICOS delivers a co-stimulatory signal that is essential both for efficient interaction between T and B cells and for normal antibody responses to T-cell-dependent antigens. To determine the physiological function of ICOS, we generated and characterized gene-targeted ICOS-deficient mice. In vivo, a lack of ICOS results in severely deficient T-cell-dependent B-cell responses. Germinal centre formation is impaired and immunoglobulin class switching, including production of allergy-mediating IgE, is defective. ICOS-deficient T cells primed in in vivo and restimulated in vitro with specific antigen produce only low levels of interleukin-4, but remain fully competent to produce interferon-gamma.     

Lysyl oxidase is essential for hypoxia-induced metastasis

Metastasis is a multistep process responsible for most cancer deaths, and it can be influenced by both the immediate microenvironment (cell-cell or cell-matrix interactions) and the extended tumour microenvironment (for example vascularization). Hypoxia (low oxygen) is clinically associated with metastasis and poor patient outcome, although the underlying processes remain unclear. Microarray studies have shown the expression of lysyl oxidase (LOX) to be elevated in hypoxic human tumour cells. Paradoxically, LOX expression is associated with both tumour suppression and tumour progression, and its role in tumorigenesis seems dependent on cellular location, cell type and transformation status. Here we show that LOX expression is regulated by hypoxia-inducible factor (HIF) and is associated with hypoxia in human breast and head and neck tumours. Patients with high LOX-expressing tumours have poor distant metastasis-free and overall survivals. Inhibition of LOX eliminates metastasis in mice with orthotopically grown breast cancer tumours. Mechanistically, secreted LOX is responsible for the invasive properties of hypoxic human cancer cells through focal adhesion kinase activity and cell to matrix adhesion. Furthermore, LOX may be required to create a niche permissive for metastatic growth. Our findings indicate that LOX is essential for hypoxia-induced metastasis and is a good therapeutic target for preventing and treating metastases.     

Mitoferrin is essential for erythroid iron assimilation

Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport and many essential redox reactions involving haemoproteins and Fe-S cluster proteins. Defective iron homeostasis results in either iron deficiency or iron overload. Precise regulation of iron transport in mitochondria is essential for haem biosynthesis, haemoglobin production and Fe-S cluster protein assembly during red cell development. Here we describe a zebrafish mutant, frascati (frs), that shows profound hypochromic anaemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake. Through positional cloning, we show that the gene mutated in the frs mutant is a member of the vertebrate mitochondrial solute carrier family (SLC25) that we call mitoferrin (mfrn). mfrn is highly expressed in fetal and adult haematopoietic tissues of zebrafish and mouse. Erythroblasts generated from murine embryonic stem cells null for Mfrn (also known as Slc25a37) show maturation arrest with severely impaired incorporation of 55Fe into haem. Disruption of the yeast mfrn orthologues, MRS3 and MRS4, causes defects in iron metabolism and mitochondrial Fe-S cluster biogenesis. Murine Mfrn rescues the defects in frs zebrafish, and zebrafish mfrn complements the yeast mutant, indicating that the function of the gene may be highly conserved. Our data show that mfrn functions as the principal mitochondrial iron importer essential for haem biosynthesis in vertebrate erythroblasts.     

Kranz anatomy is not essential for terrestrial C4 plant photosynthesis.

An important adaptation to CO2-limited photosynthesis in cyanobacteria, algae and some plants was development of CO2-concentrating mechanisms (CCM). Evolution of a CCM occurred many times in flowering plants, beginning at least 15-20 million years ago, in response to atmospheric CO2 reduction, climate change, geological trends, and evolutionary diversification of species. In plants, this is achieved through a biochemical inorganic carbon pump called C4 photosynthesis, discovered 35 years ago. C4 photosynthesis is advantageous when limitations on carbon acquisition are imposed by high temperature, drought and saline conditions. It has been thought that a specialized leaf anatomy, composed of two, distinctive photosynthetic cell types (Kranz anatomy), is required for C4 photosynthesis. We provide evidence that C4 photosynthesis can function within a single photosynthetic cell in terrestrial plants. Borszczowia aralocaspica (Chenopodiaceae) has the photosynthetic features of C4 plants, yet lacks Kranz anatomy. This species accomplishes C4 photosynthesis through spatial compartmentation of photosynthetic enzymes, and by separation of two types of chloroplasts and other organelles in distinct positions within the chlorenchyma cell cytoplasm.     

ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro

Aggrecan is the major proteoglycan in cartilage, endowing this tissue with the unique capacity to bear load and resist compression. In arthritic cartilage, aggrecan is degraded by one or more 'aggrecanases' from the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteinases. ADAMTS1, 8 and 9 have weak aggrecan-degrading activity. However, they are not thought to be the primary aggrecanases because ADAMTS1 null mice are not protected from experimental arthritis, and cleavage by ADAMTS8 and 9 is highly inefficient. Although ADAMTS4 and 5 are expressed in joint tissues, and are known to be efficient aggrecanases in vitro, the exact contribution of these two enzymes to cartilage pathology is unknown. Here we show that ADAMTS5 is the major aggrecanase in mouse cartilage, both in vitro and in a mouse model of inflammatory arthritis. Our data suggest that ADAMTS5 may be a suitable target for the development of new drugs designed to inhibit cartilage destruction in arthritis, although further work will be required to determine whether ADAMTS5 is also the major aggrecanase in human arthritis.     

The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore

A sudden increase in permeability of the inner mitochondrial membrane, the so-called mitochondrial permeability transition, is a common feature of apoptosis and is mediated by the mitochondrial permeability transition pore (mtPTP). It is thought that the mtPTP is a protein complex formed by the voltage-dependent anion channel, members of the pro- and anti-apoptotic BAX-BCL2 protein family, cyclophilin D, and the adenine nucleotide (ADP/ATP) translocators (ANTs). The latter exchange mitochondrial ATP for cytosolic ADP and have been implicated in cell death. To investigate the role of the ANTs in the mtPTP, we genetically inactivated the two isoforms of ANT in mouse liver and analysed mtPTP activation in isolated mitochondria and the induction of cell death in hepatocytes. Mitochondria lacking ANT could still be induced to undergo permeability transition, resulting in release of cytochrome c. However, more Ca2+ than usual was required to activate the mtPTP, and the pore could no longer be regulated by ANT ligands. Moreover, hepatocytes without ANT remained competent to respond to various initiators of cell death. Therefore, ANTs are non-essential structural components of the mtPTP, although they do contribute to its regulation.     

Bmi1 is essential for cerebellar development and is overexpressed in human medulloblastomas

Overexpression of the polycomb group gene Bmi1 promotes cell proliferation and induces leukaemia through repression of Cdkn2a (also known as ink4a/Arf) tumour suppressors. Conversely, loss of Bmi1 leads to haematological defects and severe progressive neurological abnormalities in which de-repression of the ink4a/Arf locus is critically implicated. Here, we show that Bmi1 is strongly expressed in proliferating cerebellar precursor cells in mice and humans. Using Bmi1-null mice we demonstrate a crucial role for Bmi1 in clonal expansion of granule cell precursors both in vivo and in vitro. Deregulated proliferation of these progenitor cells, by activation of the sonic hedgehog (Shh) pathway, leads to medulloblastoma development. We also demonstrate linked overexpression of BMI1 and patched (PTCH), suggestive of SHH pathway activation, in a substantial fraction of primary human medulloblastomas. Together with the rapid induction of Bmi1 expression on addition of Shh or on overexpression of the Shh target Gli1 in cerebellar granule cell cultures, these findings implicate BMI1 overexpression as an alternative or additive mechanism in the pathogenesis of medulloblastomas, and highlight a role for Bmi1-containing polycomb complexes in proliferation of cerebellar precursor cells.     

Sortilin is essential for proNGF-induced neuronal cell death

Sortilin (approximately 95 kDa) is a member of the recently discovered family of Vps10p-domain receptors, and is expressed in a variety of tissues, notably brain, spinal cord and muscle. It acts as a receptor for neurotensin, but predominates in regions of the nervous system that neither synthesize nor respond to this neuropeptide, suggesting that sortilin has additional roles. Sortilin is expressed during embryogenesis in areas where nerve growth factor (NGF) and its precursor, proNGF, have well-characterized effects. These neurotrophins can be released by neuronal tissues, and they regulate neuronal development through cell survival and cell death signalling. NGF regulates cell survival and cell death via binding to two different receptors, TrkA and p75NTR (ref. 10). In contrast, proNGF selectively induces apoptosis through p75NTR but not TrkA. However, not all p75NTR-expressing cells respond to proNGF, suggesting that additional membrane proteins are required for the induction of cell death. Here we report that proNGF creates a signalling complex by simultaneously binding to p75NTR and sortilin. Thus sortilin acts as a co-receptor and molecular switch governing the p75NTR-mediated pro-apoptotic signal induced by proNGF.     

MOR1 is essential for organizing cortical microtubules in plants

Microtubules orchestrate cell division and morphogenesis, but how they disassemble and reappear at different subcellular locations is unknown. Microtubule organizing centres are thought to have an important role, but in higher plants microtubules assemble in ordered configurations even though microtubule organizing centres are inconspicuous or absent. Plant cells generate highly organized microtubule arrays that coordinate mitosis, cytokinesis and expansion. Inhibiting microtubule assembly prevents chromosome separation, blocks cell division and impairs growth polarity. Microtubules are essential for the formation of cell walls, through an array of plasma-membrane-associated cortical microtubules whose control mechanisms are unknown. Using a genetic strategy to identify microtubule organizing factors in Arabidopsis thaliana, we isolated temperature-sensitive mutant alleles of the MICROTUBULE ORGANIZATION 1 (MOR1) gene. Here we show that MOR1 is the plant version of an ancient family of microtubule-associated proteins. Point mutations that substitute single amino-acid residues in an amino-terminal HEAT repeat impart reversible temperature-dependent cortical microtubule disruption, showing that MOR1 is essential for cortical microtubule organization.     

FKF1 is essential for photoperiodic-specific light signalling in Arabidopsis

Adaptation to seasonal change is a crucial component of an organism's survival strategy. To monitor seasonal variation, organisms have developed the capacity to measure day length (photoperiodism). Day-length assessment involves the photoperiodic control of flowering in Arabidopsis thaliana, whereby the coincidence of light and high expression of CONSTANS (CO) induces the expression of FLOWERING LOCUS T (FT), leading to flowering in long-day conditions. Although controlling CO expression is clearly a key step in day-length discrimination, the mechanism that generates day-length-dependent CO expression remains unknown. Here we show that the clock-controlled FLAVIN-BINDING, KELCH REPEAT, F-BOX (FKF1) protein has an essential role in generating the diurnal CO peak and that this function is dependent on light. We show that a recombinant FKF1 LIGHT, OXYGEN OR VOLTAGE (LOV) domain binds the chromophore flavin mononucleotide and undergoes light-induced photochemistry, indicating that FKF1 may function as a photoperiodic blue-light receptor. It is likely that the circadian control of FKF1 expression and the light regulation of FKF1 function coincide to control the daytime CO waveform precisely, which in turn is crucial for day-length discrimination by Arabidopsis.     

The B-cell surface protein CD72/Lyb-2 is the ligand for CD5.

The glycoprotein CD5 is expressed on the surface membrane of all mature T cells and a small proportion of B lymphocytes. Its exact role in immune interactions is still unknown. Studies indicate that CD5 functions both in mice and humans as a receptor, delivering co-stimulatory signals to T cells in a manner similar to CD2 (ref. 11) and CD28 (ref. 12). Anti-CD5 antibodies stimulate both T-cell proliferation mediated by CD3 in association with the T-cell receptor and secretion of interleukin-2 and expression of its receptor, as well as inducing an increase in intracellular Ca2+ concentration (refs 5-10). To identify the ligand for CD5 we purified the human CD5 protein, labelled it with biotin and used it as a probe. Here we report that CD5 specifically interacts with the cell-surface protein CD72 exclusive to B cells. This interaction is blocked by anti-CD72 antibodies, but not by any other anti-B-cell antibodies. Moreover, non-B cells (mouse L-cell fibroblasts and human Jurkat T cells) expressing a transfected human CD72 complementary DNA could bind to the CD5-biotin conjugate. The results demonstrate that the B-cell surface protein CD72 (Lyb-2 in mice) is the ligand for CD5.     

Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia

The vanilloid receptor-1 (VR1) is a ligand-gated, non-selective cation channel expressed predominantly by sensory neurons. VR1 responds to noxious stimuli including capsaicin, the pungent component of chilli peppers, heat and extracellular acidification, and it is able to integrate simultaneous exposure to these stimuli. These findings and research linking capsaicin with nociceptive behaviours (that is, responses to painful stimuli in animals have led to VR1 being considered as important for pain sensation. Here we have disrupted the mouse VR1 gene using standard gene targeting techniques. Small diameter dorsal root ganglion neurons isolated from VR1-null mice lacked many of the capsaicin-, acid- and heat-gated responses that have been previously well characterized in small diameter dorsal root ganglion neurons from various species. Furthermore, although the VR1-null mice appeared normal in a wide range of behavioural tests, including responses to acute noxious thermal stimuli, their ability to develop carrageenan-induced thermal hyperalgesia was completely absent. We conclude that VR1 is required for inflammatory sensitization to noxious thermal stimuli but also that alternative mechanisms are sufficient for normal sensation of noxious heat.