bluuebunny/crossref_metadata_2025 huggingface dataset:
Scabrous and Gp150 are endosomal proteins that regulate Notch activity
Authors: Li Yanxia , Fetchko Michael , Lai Zhi-Chun , Baker Nicholas E.
Published: May 2003

Notch and Delta are required for lateral inhibition during eye development. They prevent a tenfold excess in R8 photoreceptor cell specification. Mutations in two other genes, Scabrous and Gp150, result in more modestly increased R8 specification. Their roles in Notch signaling have been unclear. Both sca and gp150 are required for ectopic Notch activity that occurs in the split mutant. Similar phenotypes showed that sca and gp150 genes act in a common pathway. Gp150 was required for all activities of Sca, including inhibition of Notch activity and association with Notch-expressing cells that occur when Sca is ectopically expressed. Mosaic analysis found that the gp150 and scagenes were required in different cells from one another. Gp150 concentrated Sca protein in late endosomes. A model is proposed in which endosomal Sca and Gp150 promote Notch activation in response to Delta, by regulating acquisition of insensitivity to Delta in a subset of cells.
Somatic motoneurone specification in the hindbrain: the influence of somite-derived signals, retinoic acid and Hoxa3
Authors: Guidato Sonia , Prin Fabrice , Guthrie Sarah
Published: May 2003

We have investigated the mechanisms involved in generating hindbrain motoneurone subtypes, focusing on somatic motoneurones, which are confined to the caudal hindbrain within rhombomeres 5-8. Following heterotopic transplantation of rhombomeres along the rostrocaudal axis at various developmental stages, we have found that the capacity of rhombomeres to generate somatic motoneurones is labile at the neural plate stage but becomes fixed just after neural tube closure, at stage 10-11. Grafting of somites or retinoic acid-loaded beads beneath the rostral hindbrain induced the formation of somatic motoneurones in rhombomere 4 only, and Hox genes normally expressed more caudally (Hoxa3, Hoxd4) were induced in this region. Targeted overexpression of Hoxa3 in the rostral hindbrain led to the generation of ectopic somatic motoneurones in ventral rhombomeres 1-4, and was accompanied by the repression of the dorsoventral patterning gene Irx3. Taken together, these observations suggest that the somites,retinoic acid and Hox genes play a role in patterning somatic motoneurones in vivo.
Notch activity in neural cells triggered by a mutant allele with altered glycosylation
Authors: Li Yanxia , Li Liang , Irvine Kenneth D. , Baker Nicholas E.
Published: May 2003

The receptor protein Notch is inactive in neural precursor cells despite neighboring cells expressing ligands. We investigated specification of the R8 neural photoreceptor cells that initiate differentiation of each Drosophila ommatidium. The ligand Delta was required in R8 cells themselves, consistent with a lateral inhibitor function for Delta. By contrast, Delta expressed in cells adjacent to R8 could not activate Notch in R8 cells. The split mutation of Notch was found to activate signaling in R8 precursor cells, blocking differentiation and leading to altered development and neural cell death. split did not affect other, inductive functions of Notch. The Ile578→Thr578 substitution responsible for the split mutation introduced a new site for O-fucosylation on EGF repeat 14 of the Notch extracellular domain. The O-fucose monosaccharide did not require extension by Fringe to confer the phenotype. Our results suggest functional differences between Notch in neural and non-neural cells. R8 precursor cells are protected from lateral inhibition by Delta. The protection is affected by modifications of a particular EGF repeat in the Notch extracellular domain. These results suggest that the pattern of neurogenesis is determined by blocking Notch signaling, as well as by activating Notch signaling.
giant nucleiis essential in the cell cycle transition from meiosis to mitosis
Authors: Renault Andrew D. , Zhang Xiao-Hua , Alphey Luke S. , Frenz Lisa M. , Glover David M. , Saunders Robert D. C. , Axton J. Myles
Published: May 2003

At the transition from meiosis to cleavage mitoses, Drosophilarequires the cell cycle regulators encoded by the genes, giant nuclei(gnu), plutonium (plu) and pan gu(png). Embryos lacking Gnu protein undergo DNA replication and centrosome proliferation without chromosome condensation or mitotic segregation. We have identified the gnu gene encoding a novel phosphoprotein dephosphorylated by Protein phosphatase 1 at egg activation. Gnu is normally expressed in the nurse cells and oocyte of the ovary and is degraded during the embryonic cleavage mitoses. Ovarian death and sterility result from gnu gain of function. gnu function requires the activity of pan gu and plu.
Endoplasmic reticulum reorganizations and Ca2+ signaling in maturing and fertilized oocytes of marine protostome worms: the roles of MAPKs and MPF
Authors: Stricker Stephen A. , Smythe Toni L.
Published: May 2003

Before a proper Ca2+ response is produced at fertilization,oocytes typically undergo a maturation process during which their endoplasmic reticulum (ER) is restructured. In marine protostome worms belonging to the phylum Nemertea, the ER of maturing oocytes forms numerous distinct clusters that are about 5 μm in diameter. After fertilization, mature oocytes with such aggregates generate a normal series of Ca2+ oscillations and eventually disassemble their ER clusters at around the time that the oscillations cease. Immature oocytes, however, lack prominent ER clusters and fail to exhibit repetitive Ca2+ oscillations upon insemination,collectively suggesting that cell cycle-related changes in ER structure may play a role in Ca2+ signaling. To assess the effects of meiotic regulators on the morphology of the ER and the type of Ca2+response that is produced at fertilization, nemertean oocytes were treated with pharmacological modulators of mitogen-activated protein kinases (MAPKs)or maturation-promoting factor (MPF) prior to confocal microscopic analyses. Based on such imaging studies and correlative assays of kinase activities,MAPKs of the ERK1/2 type (extracellular signal regulated kinases 1/2) do not seem to be essential for either structural reorganizations of the ER or repetitive Ca2+ signaling at fertilization. Conversely, MPF levels appear to modulate both ER structure and the capacity to produce normal Ca2+ oscillations. The significance of these findings is discussed with respect to other reports on ER structure, MPF cycling and Ca2+signaling in oocytes of deuterostome animals.
Requirement for two nearly identical TGIF-related homeobox genes in Drosophila spermatogenesis
Authors: Wang Zhaohui , Mann Richard S.
Published: May 2003

The genetic analysis of spermatogenesis in Drosophila melanogasterhas led to the identification of several genes that control the onset of meiosis, spermatid differentiation, or both. We described two tightly linked and nearly identical homeobox genes of the TGIF (TG-interacting factor)subclass called vismay and achintya that are essential for spermatogenesis in Drosophila. In flies deficient for both genes,spermatogenesis is blocked prior to any spermatid differentiation and before the first meiotic division. This suggests that vismay and achintya function at the same step as two previously characterized meiotic arrest genes, always early and cookie monster. Consistent with this idea, both always early and cookie monster are still expressed in flies deficient in vismay and achintya. Conversely, Vismay and Achintya proteins are present in always early mutant testes. Co-immunoprecipitation experiments further suggest that Vismay and Achintya proteins exist in a complex with Always early and Cookie monster proteins. Because Vismay and Achintya are likely to be sequence-specific DNA binding factors, these results suggest that they help to specify the spermatogenesis program by recruiting or stabilizing Always early and Cookie monster to specific target genes that need to be transcriptionally regulated during testes development.
Alx1, a member of the Cart1/Alx3/Alx4 subfamily of Paired-class homeodomain proteins, is an essential component of the gene network controlling skeletogenic fate specification in the sea urchin embryo
Authors: Ettensohn Charles A. , Illies Michele R. , Oliveri Paola , De Jong Deborah L.
Published: May 2003

In the sea urchin embryo, the large micromeres and their progeny function as a critical signaling center and execute a complex morphogenetic program. We have identified a new and essential component of the gene network that controls large micromere specification, the homeodomain protein Alx1. Alx1 is expressed exclusively by cells of the large micromere lineage beginning in the first interphase after the large micromeres are born. Morpholino studies demonstrate that Alx1 is essential at an early stage of specification and controls downstream genes required for epithelial-mesenchymal transition and biomineralization. Expression of Alx1 is cell autonomous and regulated maternally through β-catenin and its downstream effector, Pmar1. Alx1 expression can be activated in other cell lineages at much later stages of development, however, through a regulative pathway of skeletogenesis that is responsive to cell signaling. The Alx1 protein is highly conserved among euechinoid sea urchins and is closely related to the Cart1/Alx3/Alx4 family of vertebrate homeodomain proteins. In vertebrates, these proteins regulate the formation of skeletal elements of the limbs, face and neck. Our findings suggest that the ancestral deuterostome had a population of biomineral-forming mesenchyme cells that expressed an Alx1-like protein.
DrosophilaTGIF is essential for developmentally regulated transcription in spermatogenesis
Authors: Ayyar Savita , Jiang Jianqiao , Collu Anna , White-Cooper Helen , White Robert A. H.
Published: May 2003

We have investigated the role of TGIF, a TALE-class homeodomain transcription factor, in Drosophila development. In vertebrates, TGIF has been implicated, by in vitro analysis, in several pathways, most notably as a repressor modulating the response to TGFβ signalling. Human TGIF has been associated with the developmental disorder holoprosencephaly. Drosophila TGIF is represented by the products of two tandemly repeated highly similar genes, achintya and vismay. We have generated mutations that delete both genes. Homozygous mutant flies are viable and appear morphologically normal, but the males are completely sterile. The defect lies at the primary spermatocyte stage and differentiation is blocked prior to the onset of the meiotic divisions. We show that mutants lacking TGIF function fail to activate transcription of many genes required for sperm manufacture and of some genes required for entry into the meiotic divisions. This groups TGIF together with two other genes producing similar phenotypes, always early and cookie monster, as components of the machinery required for the activation of the spermatogenic programme of transcription. TGIF is the first sequence-specific transcription factor identified in this pathway. By immunolabelling in mouse testes we show that TGIF is expressed in the early stages of spermatogenesis consistent with a conserved role in the activation of the spermatogenesis transcription programme.
The glial sling is a migratory population of developing neurons
Authors: Shu Tianzhi , Li Ying , Keller Asaf , Richards Linda J.
Published: May 2003

For two decades the glial sling has been hypothesized to act as a guidance substratum for developing callosal axons. However, neither the cellular nature of the sling nor its guidance properties have ever been clearly identified. Although originally thought to be glioblasts, we show here that the subventricular zone cells forming the sling are in fact neurons. Sling cells label with a number of neuronal markers and display electrophysiological properties characteristic of neurons and not glia. Furthermore, sling cells are continuously generated until early postnatal stages and do not appear to undergo widespread cell death. These data indicate that the sling may be a source of, or migratory pathway for, developing neurons in the rostral forebrain, suggesting additional functions for the sling independent of callosal axon guidance.
Retinal ganglion cell-derived sonic hedgehog signaling is required for optic disc and stalk neuroepithelial cell development
Authors: Dakubo Gabriel D. , Wang Ya Ping , Mazerolle Chantal , Campsall Katrina , McMahon Andrew P. , Wallace Valerie A.
Published: May 2003

The development of optic stalk neuroepithelial cells depends on Hedgehog(Hh) signaling, yet the source(s) of Hh protein in the optic stalk is unknown. We provide genetic evidence that sonic hedgehog (Shh) from retinal ganglion cells (RGCs) promotes the development of optic disc and stalk neuroepithelial cells. We demonstrate that RGCs express Shh soon after differentiation, and cells at the optic disc in close proximity to the Shh-expressing RGCs upregulate Hh target genes, which suggests they are responding to RGC-derived Shh signaling. Conditional ablation of Shhin RGCs caused a complete loss of optic disc astrocyte precursor cells,resulting in defective axon guidance in the retina, as well as conversion of the neuroepithelial cells in the optic stalk to pigmented cells. We further show that Shh signaling modulates the size of the Pax2+ astrocyte precursor cell population at the optic disc in vitro. Together, these data provide a novel insight into the source of Hh that promotes neuroepithelial cell development in the mammalian optic disc and stalk.
Gata factor Pannier is required to establish competence for heart progenitor formation
Authors: Klinedinst Susan L. , Bodmer Rolf
Published: May 2003

Inductive signaling is of pivotal importance for developmental patterns to form. In Drosophila, the transfer of TGFβ (Dpp) and Wnt (Wg)signaling information from the ectoderm to the underlying mesoderm induces cardiac-specific differentiation in the presence of Tinman, a mesoderm-specific homeobox transcription factor. We present evidence that the Gata transcription factor, Pannier, and its binding partner U-shaped, also a zinc-finger protein, cooperate in the process of heart development. Loss-of-function and germ layer-specific rescue experiments suggest that pannier provides an essential function in the mesoderm for initiation of cardiac-specific expression of tinman and for specification of the heart primordium. u-shaped also promotes heart development, but unlike pannier, only by maintaining tinman expression in the cardiogenic region. By contrast, pan-mesodermal overexpression of pannier ectopically expands tinman expression, whereas overexpression of u-shaped inhibits cardiogenesis. Both factors are also required for maintaining dpp expression after germ band retraction in the dorsal ectoderm. Thus, we propose that Pannier mediates as well as maintains the cardiogenic Dpp signal. In support, we find that manipulation of pannier activity in either germ layer affects cardiac specification, suggesting that its function is required in both the mesoderm and the ectoderm.
Two Pax genes,eye goneandeyeless, act cooperatively in promotingDrosophilaeye development
Authors: Jang Chuen-Chuen , Chao Ju-Lan , Jones Nikolas , Yao Li-Chin , Bessarab Dmitri A. , Kuo Yien M. , Jun Susie , Desplan Claude , Beckendorf Steven K. , Sun Y. Henry
Published: May 2003

We report the identification of a Drosophila Pax gene, eye gone (eyg), which is required for eye development. Loss-of-function eyg mutations cause reduction or absence of the eye. Similar to the Pax6 eyeless (ey) gene, ectopic expression of eyg induces extra eye formation, but at sites different from those induced by ey. Several lines of evidence suggest that eygand ey act cooperatively: (1) eyg expression is not regulated by ey, nor does it regulate ey expression, (2) eyg-induced ectopic morphogenetic furrow formation does not require ey, nor does ey-induced ectopic eye production require eyg, (3) eyg and ey can partially substitute for the function of the other, and (4) coexpression of eyg and ey has a synergistic enhancement of ectopic eye formation. Our results also show that eyg has two major functions: to promote cell proliferation in the eye disc and to promote eye development through suppression of wg transcription.
TagA, a putative serine protease/ABC transporter ofDictyosteliumthat is required for cell fate determination at the onset of development
Authors: Good J. Randall , Cabral Matthew , Sharma Sujata , Yang Jun , Van Driessche Nancy , Shaw Chad A. , Shaulsky Gad , Kuspa Adam
Published: May 2003

The tag genes of Dictyostelium are predicted to encode multi-domain proteins consisting of serine protease and ATP-binding cassette transporter domains. We have identified a novel tag gene, tagA, which is involved in cell type differentiation. The tagA mRNA accumulates during the first four hours of development,whereas TagA protein accumulates between two and ten hours of development and decreases thereafter. Wild-type cells express tagA in prespore cells and mature spores, defining tagA expression as prespore specific. However, tagA mutant cells that activate the tagA promoter do not sporulate, but instead form part of the outer basal disc and lower cup of the fruiting body. tagA mutant aggregates elaborate multiple prestalk cell regions during development and produce spores asynchronously and with low viability. tagA mutants produce about twice as many prestalk cells as the wild type as judged by a prestalk cell reporter construct. When mixed with wild-type cells, tagA- cells become overrepresented in the prestalk cell population, suggesting that this phenotype is cell-autonomous. These results suggest that TagA is required for the specification of an initial population of prespore cells in which tagA is expressed. Expression profiling uncovered a delay in the transcriptional program between 2 and 6 hours, coincident with TagA expression, revealing an early function for TagA. TagA also appears to play a general role in cell fate determination since tagA mutants express a spore coat protein gene (cotB) within vacuolated cells that form part of the stalk and they express a prestalk/stalk-specific gene (ecmB)within cells that become spores. The expression of TagA at two hours of development, the observed coincident delay in the transcriptional program and the subsequent mis-expression of cell-type specific genes provide evidence for cell fate determination beginning in some cells much earlier than previously believed.
Strabismus is asymmetrically localised and binds to Prickle and Dishevelled duringDrosophilaplanar polarity patterning
Authors: Bastock Rebecca , Strutt Helen , Strutt David
Published: May 2003

Planar polarity decisions in the wing of Drosophila involve the assembly of asymmetric protein complexes containing the conserved receptor Frizzled. In this study, we analyse the role of the Van Gogh/strabismus gene in the formation of these complexes and cell polarisation. We find that the Strabismus protein becomes asymmetrically localised to the proximal edge of cells. In the absence of strabismusactivity, the planar polarity proteins Dishevelled and Prickle are mislocalised in the cell. We show that Strabismus binds directly to Dishevelled and Prickle and is able to recruit them to membranes. Furthermore,we demonstrate that the putative PDZ-binding motif at the C terminus of Strabismus is not required for its function. We propose a two-step model for assembly of Frizzledcontaining asymmetric protein complexes at cell boundaries. First, Strabismus acts together with Frizzled and the atypical cadherin Flamingo to mediate apicolateral recruitment of planar polarity proteins including Dishevelled and Prickle. In the second phase, Dishevelled and Prickle are required for these proteins to become asymmetrically distributed on the proximodistal axis.
Mechanism ofhedgehogsignaling duringDrosophilaeye development
Authors: Pappu Kartik S. , Chen Rui , Middlebrooks Brooke W. , Woo Catherine , Heberlein Ulrike , Mardon Graeme
Published: May 2003

Although Hedgehog (Hh) signaling is essential for morphogenesis of the Drosophila eye, its exact link to the network of tissue-specific genes that regulate retinal determination has remained elusive. In this report, we demonstrate that the retinal determination gene eyes absent (eya) is the crucial link between the Hedgehog signaling pathway and photoreceptor differentiation. Specifically, we show that the mechanism by which Hh signaling controls initiation of photoreceptor differentiation is to alleviate repression of eya and decapentaplegic (dpp) expression by the zinc-finger transcription factor Cubitus interruptus (Cirep). Furthermore, our results suggest that stabilized, full length Ci (Ciact) plays little or no role in Drosophila eye development. Moreover, while the effects of Hh are primarily concentration dependent in other tissues, hh signaling in the eye acts as a binary switch to initiate retinal morphogenesis by inducing expression of the tissue-specific factor Eya.
Ectopic expression of syntaxin 1 in the ER redirects TI-VAMP- and cellubrevin-containing vesicles
Authors: Martinez-Arca Sonia , Proux-Gillardeaux Véronique , Alberts Philipp , Louvard Daniel , Galli Thierry
Published: May 2003

SNARE proteins are key mediators of membrane fusion. Their function in ensuring compartmental specificity of membrane fusion has been suggested by in vitro studies but not demonstrated in vivo. We show here that ectopic expression of the plasma membrane t-SNARE heavy chain syntaxin 1 in the endoplasmic reticulum induces the redistribution of its cognate vesicular SNAREs, TI-VAMP and cellubrevin, and its light chain t-SNARE SNAP-23. These effects were prevented by co-expressing nSec1. Expression of syntaxin 1 alone impaired the cell surface expression of TI-VAMP and cellubrevin but not the recycling of transferrin receptor. TI-VAMP, cellubrevin and SNAP-23 associated in vivo with exogenous syntaxin 1. Redistribution of TI-VAMP in the ER of syntaxin-1-expressing cells was microtubule dependent and impaired the trafficking of CD63, a cargo of TI-VAMP-containing vesicles. We conclude that the destination of v-SNAREs is driven by their specific interaction with cognate t-SNAREs. Our in vivo data provide strong support for the theory that highly specific v-SNARE–t-SNARE interactions control compartmental specificity of membrane fusion.
Pollen tubes exhibit regular periodic membrane trafficking events in the absence of apical extension
Authors: Parton Richard M. , Fischer-Parton Sabine , Trewavas Anthony J. , Watahiki Masaaki K.
Published: May 2003

The growing pollen tube provides an excellent single cell model system in which to study the mechanisms determining growth regulation, polarity and periodic behaviour. Previously, using FM4-64, we identified periodic movements within the apical vesicle accumulation that were related to the period of oscillatory growth. This suggested a more complex interdependence between membrane traffic, apical extension and periodicity than previously thought. To investigate this a comparison was made between normally growing and Brefeldin-A-treated, non-growing, tubes. Brefeldin-A treatment established an intriguing, stable yet dynamic system of membrane aggregations in the pollen tube tip that exhibited regular movements of material with a 5-7 second period compared with the normal ∼30 second periodicity observed in growing tubes. Heat treatment was found to reduce period length in both cases. After BFA treatment membrane was demonstrated to flow from the extreme pollen tube apex back through a distinct subapical Brefeldin-A-induced membrane accumulation. The effects of Brefeldin-A on the distribution of ER- and Golgi-targeted fluorescent proteins revealed that ER did not contribute directly to the system of membrane aggregations while only certain compartments of the Golgi might be involved. The involvement of membrane derived from the apical vesicle accumulation was strongly implicated. Calcium measurements revealed that Brefeldin-A abolished the typical tip-focused calcium gradient associated with growth and there were no obvious periodic fluctuations in apical calcium associated with the continued periodic Brefeldin-A membrane aggregation associated movements. Our experiments reveal an underlying periodicity in the pollen tube that is independent of secretion, apical extension and the oscillating tip-focused calcium gradient normally associated with growth, but requires an active actin cytoskeleton.
A 48 kDa integral membrane phosphoprotein orchestrates the cytoskeletal dynamics that generate amoeboid cell motility in Ascaris sperm
Authors: LeClaire Lawrence L. , Stewart Murray , Roberts Thomas M.
Published: May 2003

Protrusion of the lamellipod in the crawling sperm of Ascaris is tightly coupled to the localized vectorial assembly and bundling of the major sperm protein cytoskeleton. In cell-free extracts of sperm, vesicles derived from the leading edge membrane reconstitute protrusion by directing the assembly of columnar meshworks of major sperm protein filaments that push the vesicle forward as they elongate. Treatment with proteases or a tyrosine phosphatase abolished vesicle activity, suggesting the involvement of a membrane phosphoprotein. Fractionation of vesicle proteins by sequential detergent lysis, size exclusion chromatography and immunoprecipitation with antiphosphotyrosine antibody identified a 48 kDa integral membrane phosphoprotein as the only sperm membrane component required to nucleate major sperm protein polymerization under physiological conditions. Immunolabeling assays showed that this protein is distributed uniformly in the sperm plasma membrane, but that its active phosphorylated form is located only at sites of major sperm protein polymerization at the leading edge. Because this protein specifies sites of cytoskeletal assembly, we have named it major sperm protein polymerization organizing protein (MPOP). The phosphorylation of MPOP is pH sensitive and appears to require a soluble tyrosine kinase. Comparison of the activity of MPOP to that of analogous membrane proteins in actin-based systems emphasizes the importance of precise transmission of information from the membrane to the cytoskeleton in amoeboid cell motility.
CD105 prevents apoptosis in hypoxic endothelial cells
Authors: Li Chenggang , Issa Razao , Kumar Pat , Hampson Ian N. , Lopez-Novoa Jose M. , Bernabeu Carmelo , Kumar Shant
Published: May 2003

CD105, a marker of endothelial cells, is abundantly expressed in tissues undergoing angiogenesis and is a receptor for transforming growth factorβ. The pivotal role of CD105 in the vascular system was demonstrated by the severe vascular defects that occur in CD105-knockout mice,but the exact mechanisms for CD105 regulation of vascular development have not been fully elucidated. In light of the function of CD105 and the importance of hypoxia in neovascularisation, we speculated that CD105 is involved in hypoxia-initiated angiogenesis. Using tissue-cultured human microvascular endothelial cells, we have investigated the effects of hypoxic stress on CD105 gene expression. Hypoxia induced a significant increase in membrane-bound and secreted CD105 protein levels. CD105 mRNA and promoter activity were also markedly elevated, the latter returning to the basal level after 16 hours of hypoxic stress. Hypoxia induced cell cycle arrest at the G0/G1 phases and massive cell apoptosis after 24 hours through a reduction in the Bcl-2 to Bax ratio, downregulation of Bcl-XL and Mcl-1, and upregulation of caspase-3 and caspase-8. The consequence of CD105 upregulation was revealed using an antisense approach and a TUNEL assay. Suppression of CD105 increased cell apoptosis under hypoxic stress in the absence of TGFβ1. Furthermore,hypoxia and TGFβ1 synergistically induced apoptosis in the CD105-deficient cells but not in the control cells. We conclude that hypoxia is a potent stimulus for CD105 gene expression in vascular endothelial cells,which in turn attenuates cell apoptosis and thus contributes to angiogenesis.
S100A13 mediates the copper-dependent stress-induced release of IL-1α from both human U937 and murine NIH 3T3 cells
Authors: Mandinova Anna , Soldi Raffaella , Graziani Irene , Bagalá Cinzia , Bellum Stephen , Landriscina Matteo , Tarantini Francesca , Prudovsky Igor , Maciag Thomas
Published: May 2003

Copper is involved in the promotion of angiogenic and inflammatory events in vivo and, although recent clinical data has demonstrated the potential of Cu2+ chelators for the treatment of cancer in man, the mechanism for this activity remains unknown. We have previously demonstrated that the signal peptide-less angiogenic polypeptide, FGF1, uses intracellular Cu2+ to facilitate the formation of a multiprotein aggregate that enables the release of FGF1 in response to stress and that the expression of the precursor form but not the mature form of IL-1α represses the stress-induced export of FGF1 from NIH 3T3 cells. We report here that IL-1α is a Cu2+-binding protein and human U937 cells, like NIH 3T3 cells, release IL-1α in response to temperature stress in a Cu2+-dependent manner. We also report that the stress-induced export of IL-1α involves the intracellular association with the Cu2+-binding protein, S100A13. In addition, the expression of a S100A13 mutant lacking a sequence novel to this gene product functions as a dominant-negative repressor of IL-1α release, whereas the expression of wild-type S100A13 functions to eliminate the requirement for stress-induced transcription. Lastly, we present biophysical evidence that IL-1α may be endowed with molten globule character, which may facilitate its release through the plasma membrane. Because Cu2+ chelation also represses the release of FGF1, the ability of Cu2+ chelators to potentially serve as effective clinical anti-cancer agents may be related to their ability to limit the export of these proinflammatory and angiogenic signal peptide-less polypeptides into the extracellular compartment.
Unexpected roles of aDictyosteliumhomologue of eukaryotic EF-2 in growth and differentiation
Authors: Watanabe Sohsuke , Sakurai Kohji , Amagai Aiko , Maeda Yasuo
Published: May 2003

EF-2 is believed to be indispensable for polypeptide chain elongation in protein synthesis and therefore for cell proliferation. Surprisingly, we could isolate ef2 null cells from Dictyostelium discoideum that exhibited almost normal growth and protein synthesis, which suggests that there is another molecule capable of compensating for EF-2 function. The knock-out of Dictyostelium EF-2 (Dd-EF2H; 101 kDa phosphoprotein)impairs cytokinesis, resulting in formation of multinucleate cells. The initiation of differentiation, including the acquisition of aggregation competence, was delayed in Dd-ef2 null cells compared with that in wild-type. By contrast, Dd-ef2 overexpression enhanced the progression of differentiation, thus indicating a positive involvement of Dd-EF2H in growth/differentiation transition.
Regulation of EDEN-dependent deadenylation of Aurora A/Eg2-derived mRNA via phosphorylation and dephosphorylation inXenopus laevisegg extracts
Authors: Detivaud Lénaïck , Pascreau Gaëtan , Karaïskou Anthi , Osborne Howard B. , Kubiak Jacek Z.
Published: May 2003

Deadenylation is an intimate part of the post-transcriptional regulation of maternal mRNAs in embryos. EDEN-BP is so far the only known member of a complex regulating the deadenylation of maternal mRNA in Xenopus laevis embryos in a manner that is dependent on the 3′-untranslated region called EDEN (embryo deadenylation element). In this report, we show that calcium activation of cell-free extracts triggers EDEN binding protein(EDEN-BP) dephosphorylation and concomitant deadenylation of a chimeric RNA bearing Aurora A/Eg2 EDEN sequence. Deadenylation of mRNA deprived of EDEN sequence (default deadenylation) does not change with egg activation. Kinase and phosphatase inhibitors downregulate EDEN-dependent deadenylation but they do not substantially influence default deadenylation. Using indestructibleΔ90 cyclin B to revert interphase extracts to the M-phase, we show that modulation of EDEN-dependent deadenylation is independent of M-phase promoting factor (MPF) activity. These results suggest that the increase in EDEN-dependent deadenylation following egg activation is achieved, at least partially, via dephosphorylation and/or phosphorylation of regulatory proteins, including EDEN-BP dephosphorylation. This regulation proceeds in a manner independent from MPF inactivation.
Glutamate-mediated [Ca2+]c dynamics in spontaneously firing dopamine neurons of the rat substantia nigra pars compacta
Authors: Choi Yu Mi , Kim Shin Hye , Uhm Dae Yong , Park Myoung Kyu
Published: May 2003

The mechanism by which glutamate regulates the cytosolic free Ca2+ concentration ([Ca2+]c) in spontaneously firing dopamine neurons is not clear. Thus we have investigated the glutamate-mediated [Ca2+]c dynamics in the acutely isolated dopamine neurons from the rat substantia nigra pars compacta by measuring [Ca2+]c and spontaneously occurring action potentials (SAPs). The freshly isolated dopamine neurons showed tetrodotoxin(TTX)-sensitive spontaneous firing of 2-3 Hz and the resting[Ca2+]c decreased with abolition of the SAPs. The level of [Ca2+]c was affected by the spontaneous firing rate. In the presence of the Na+ channel antagonist, TTX (0.5 μM),glutamate increased [Ca2+]c by activating different glutamate receptors depending on the glutamate concentration used. Addition of glutamate at low concentrations (<3 μM) raised[Ca2+]c mainly by activating metabotropic glutamate receptors (mGluR), whereas at high concentrations (>10 μM) it raised[Ca2+]c mainly by activating AMPA/kainate receptors. The contribution of NMDA receptors to the glutamate-mediated[Ca2+]c rises was largest at intermediate concentrations of glutamate. Activation of mGluR elicited a Ca2+ release from intracellular Ca2+ stores and continuous Ca2+ influx out of the cell. The spontaneous firing activities were highly enhanced by submicromolar levels of glutamate and abolished at levels above 10 μM. From these results, we conclude that at low glutamate concentrations the[Ca2+]c in the dopamine neurons is mainly governed by mGluR and the firing activities, whose rate is regulated at submicromolar glutamate concentrations, but at higher glutamate concentrations[Ca2+]c is dominantly affected by AMPA/kainate receptors.
Centrin deficiency inChlamydomonascauses defects in basal body replication, segregation and maturation
Authors: Koblenz Bettina , Schoppmeier Jutta , Grunow Andrea , Lechtreck Karl-Ferdinand
Published: May 2003

Centrin, a 20 kDa calcium-binding protein, is a constituent of contractile basal body-associated fibers in protists and of various centrosomal structures. A construct inducing centrin RNAi was used to study the effect of centrin deficiency in Chlamydomonas. Transformants contained variable amounts of residual centrin (down to 5% of wild-type) and lacked centrin fibers. They displayed a variable flagellar number phenotype with mostly nonflagellate cells, suggesting that centrin is required for basal body assembly. Furthermore, basal bodies often failed to dock to the plasma membrane and to assemble flagella, and displayed defects in the flagellar root system indicating that centrin deficiency interferes with basal body development. Multiple basal bodies caused the formation of additional microtubular asters, whereas the microtubular cytoskeleton was disordered in most cells without basal bodies. The number of multinucleated cells was increased, indicating that aberrant numbers of basal bodies interfered with the cytokinesis of Chlamydomonas. In contrast to wild-type cells,basal bodies in centrin-RNAi cells were separated from the spindle poles,suggesting a role of centrin in tethering basal bodies to the spindle. To test whether an association with the spindle poles is required for correct basal body segregation, we disrupted centrin fibers in wild-type cells by over-expressing a nonfunctional centrin-GFP. In these cells, basal bodies were disconnected from the spindle but segregation errors were not observed. We propose that basal body segregation in Chlamydomonas depends on an extranuclear array of microtubules independent of the mitotic spindle.
Actin-based photo-orientation movement of chloroplasts in plant cells
Authors: Takagi Shingo
Published: May 2003

In photosynthesizing plant cells, chloroplasts change their arrangements and/or positions in response to light irradiation. These photo-orientation movements of chloroplasts are believed to play important roles in optimizing the photosynthetic activity of plant cells. We have been investigating the roles of the actin cytoskeleton in the intracellular movement and positioning of chloroplasts using the aquatic monocot Vallisneria giganteaGraebner and the terrestrial dicot Spinacia oleracea L. (spinach). In Vallisneria epidermal cells, chloroplasts accumulate on the cytoplasmic layer facing the top surface (outer periclinal layer) under dim red light, whereas they move to the cytoplasmic layer perpendicular to the outer periclinal layer (anticlinal layer) under strong blue light. Concomitant with these responses, actin filaments exhibit dramatic changes in their configurations. The possible modes of action of the actin cytoskeleton to regulate the movement and positioning of chloroplasts are briefly summarized,together with our recent analysis of the association of actin filaments with chloroplasts isolated from spinach leaves.
Diffusion, perfusion and the exclusion principles in the structural and functional organization of the living cell: reappraisal of the properties of the `ground substance'
Authors: Wheatley Denys N.
Published: May 2003

The thesis is presented that only within very small microdomains of the cell internum might diffusion operate in the sorting of molecular affinities. Much of cell metabolism is guided and controlled in rate by the speed with which molecules that have to interact encounter one another. What is clear,however, is that the cell does not have a single `modus operandi' but has the choice of many different strategies, each of which can contribute in different proportion to the rate of ongoing activity. It is probably our own desire to simplify things and use the most (or more) probable strategy that confines our appreciation of the overall robustness of the cell as a `survival machine'. The main operative process at any given time (perfusion, diffusion or whatever) has always to be considered very carefully in relation to the organisational structure of the cell, which can be transient and fickle but nevertheless has been seen as involving an extensive cytomatrix, a ground substance, within an aqueous environment in which the degree of water structuring is even more fickle.
Force–velocity relationships in actin–myosin interactions causing cytoplasmic streaming in algal cells
Authors: Sugi Haruo , Chaen Shigeru
Published: May 2003

Cytoplasmic streaming in giant internodal cells of green algae is caused by ATP-dependent sliding between actin cables fixed on chloroplast rows and cytoplasmic myosin molecules attached to cytoplasmic organelles. Its velocity(≥50 μm s-1) is many times larger than the maximum velocity of actin–myosin sliding in muscle. We studied kinetic properties of actin–myosin sliding causing cytoplasmic streaming in internodal cell preparations of Chara corallina, into which polystyrene beads, coated with cytoplasmic myosin molecules, were introduced. Constant centrifugal forces directed opposite to the bead movement were applied as external loads. The steady-state force–velocity(P–V) curves obtained were nearly straight, irrespective of the maximum isometric force generated by cytoplasmic myosin molecules, indicating a large duty ratio of cytoplasmic myosin head. The large velocity of cytoplasmic streaming can be accounted for, at least qualitatively, by assuming a mechanically coupled interaction between cytoplasmic myosin heads as well as a large distance of unitary actin–myosin sliding.
Actin comet tails, endosomes and endosymbionts
Authors: Fehrenbacher Kammy , Huckaba Thomas , Yang Hyeong-Cheol , Boldogh Istvan , Pon Liza
Published: May 2003

The Arp2/3 complex consists of seven highly conserved and tightly associated subunits, two of which are the actin-related proteins Arp2 and Arp3. One of the best-studied functions of the Arp2/3 complex is to stimulate actin nucleation and force production at the leading edge of motile cells. What is now clear is that Arp2/3-complex-mediated force production drives many intracellular movements, including movement of bacterial pathogens in infected host cells, internalization of extracellular materials viaphagocytosis and endocytosis, and movement of mitochondria during cell division in budding yeast. Here, we describe recent advances in the mechanisms underlying Arp2/3 complex-driven intracellular movement.
Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function
Authors: Wilson John E.
Published: May 2003

The first step in metabolism of glucose (Glc) is usually phosphorylation,catalyzed by hexokinase. However, the Glc-6-P produced can then enter one or more of several alternative pathways. Selective expression of isozymic forms of hexokinase, differing in catalytic and regulatory properties as well as subcellular localization, is likely to be an important factor in determining the pattern of Glc metabolism in mammalian tissues/cells. Despite their overall structural similarity, the Type I, Type II and Type III isozymes differ in important respects. All three isozymes are inhibited by the product,Glc-6-P, but with the Type I isozyme, this inhibition is antagonized by PI, whereas with the Type II and Type III isozymes, Piactually causes additional inhibition. Reciprocal changes in intracellular levels of Glc-6-P and Pi are closely associated with cellular energy status, and it is proposed that the response of the Type I isozyme to these effectors adapts it for catabolic function, introducing Glc into glycolytic metabolism for energy production. In contrast, the Type II, and probably the Type III, isozymes are suggested to serve primarily anabolic functions, e.g. to provide Glc-6-P for glycogen synthesis or metabolism via the pentose phosphate pathway for lipid synthesis. Type I hexokinase binds to mitochondria through interaction with porin, the protein that forms channels through which metabolites traverse the outer mitochondrial membrane. Several experimental approaches have led to the conclusion that the Type I isozyme, bound to actively phosphorylating mitochondria, selectively uses intramitochondrial ATP as substrate. Such interactions are thought to facilitate coordination of the introduction of Glc into glycolysis, via the hexokinase reaction, with the terminal oxidative stages of Glc metabolism occurring in the mitochondria, thus ensuring an overall rate of Glc metabolism commensurate with cellular energy demands and avoiding excessive production of lactate. The Type II isozyme also binds to mitochondria. Whether such coupling occurs with mitochondrially bound Type II hexokinase in normal tissues, and how it might be related to the proposed anabolic role of this isozyme, remain to be determined. The Type III isozyme lacks the hydrophobic N-terminal sequence known to be critical for binding of the Type I and Type II isozymes to mitochondria. Immunolocalization studies have indicated that, in many cell types, the Type III has a perinuclear localization, the possible metabolic consequences of which remain unclear.
Metabolic consequences of functional complexes of mitochondria,myofibrils and sarcoplasmic reticulum in muscle cells
Authors: Andrienko T. , Kuznetsov A. V. , Kaambre T. , Usson Y. , Orosco A. , Appaix F. , Tiivel T. , Sikk P. , Vendelin M. , Margreiter R. , Saks V. A.
Published: May 2003

Regulation of mitochondrial respiration both by endogenous and exogenous ADP in the cells in situ was studied in isolated and permeabilized cardiomyocytes, permeabilized cardiac fibers and `ghost' fibers (all with a diameter of 10–20 μm) at different (0–3 μmoll-1)free Ca2+ concentrations in the medium. In all these preparations,the apparent Km of mitochondrial respiration for exogenous ADP at free Ca2+ concentrations of 0–0.1μmoll-1 was very high, in the range of 250–350μmoll-1, in contrast to isolated mitochondria in vitro(apparent Km for ADP is approximately 20μmoll-1). An increase in the free Ca2+ concentration(up to 3 μmoll-1, which is within physiological range), resulted in a very significant decrease of the apparent Km value to 20–30 μmoll-1, a decrease of Vmax of respiration in permeabilized intact fibers and a strong contraction of sarcomeres. In ghost cardiac fibers, from which myosin was extracted but mitochondria were intact, neither the high apparent Km for ADP (300–350 μmoll-1) nor Vmax of respiration changed in the range of free Ca2+ concentration studied, and no sarcomere contraction was observed. The exogenous-ADP-trapping system (pyruvate kinase + phosphoenolpyruvate) inhibited endogenous-ADP-supported respiration in permeabilized cells by no more than 40%, and this inhibition was reversed by creatine due to activation of mitochondrial creatine kinase. These results are taken to show strong structural associations (functional complexes) among mitochondria, sarcomeres and sarcoplasmic reticulum. Inside these complexes, mitochondrial functional state is controlled by channeling of ADP, mostly via energy- and phosphoryl-transfer networks, and apparently depends on the state of sarcomere structures.
Myoglobin function reassessed
Authors: Wittenberg Jonathan B. , Wittenberg Beatrice A.
Published: May 2003

The heart and those striated muscles that contract for long periods, having available almost limitless oxygen, operate in sustained steady states of low sarcoplasmic oxygen pressure that resist change in response to changing muscle work or oxygen supply. Most of the oxygen pressure drop from the erythrocyte to the mitochondrion occurs across the capillary wall. Within the sarcoplasm,myoglobin, a mobile carrier of oxygen, is developed in response to mitochondrial demand and augments the flow of oxygen to the mitochondria. Myoglobin-facilitated oxygen diffusion, perhaps by virtue of reduction of dimensionality of diffusion from three dimensions towards two dimensions in the narrow spaces available between mitochondria, is rapid relative to other parameters of cell respiration. Consequently, intracellular gradients of oxygen pressure are shallow, and sarcoplasmic oxygen pressure is nearly the same everywhere. Sarcoplasmic oxygen pressure, buffered near 0.33 kPa (2.5 torr; equivalent to approximately 4 μmol l-1 oxygen) by equilibrium with myoglobin, falls close to the operational Km of cytochrome oxidase for oxygen, and any small increment in sarcoplasmic oxygen pressure will be countered by increased oxygen utilization. The concentration of nitric oxide within the myocyte results from a balance of endogenous synthesis and removal by oxymyoglobin-catalyzed dioxygenation to the innocuous nitrate. Oxymyoglobin,by controlling sarcoplasmic nitric oxide concentration, helps assure the steady state in which inflow of oxygen into the myocyte equals the rate of oxygen consumption.