During Drosophila embryogenesis the two halves of the lateral epidermis migrate dorsally over a s... more During Drosophila embryogenesis the two halves of the lateral epidermis migrate dorsally over a surface of flattened cells, the amnioserosa, and meet at the dorsal midline in order to form the continuous sheet of the larval epidermis. During this process of epithelial migration, known as dorsal closure, signaling from a Jun-amino-terminal-kinase cascade causes the production of the secreted transforming-growth-factor-beta-like ligand, Decapentaplegic. Binding of Decapentaplegic to the putative transforming-growth-factor-beta-like receptors Thickveins and Punt activates a transforming-growth-factor-beta-like pathway that is also required for dorsal closure. Mutations in genes involved in either the Jun-amino-terminal-kinase cascade or the transforming-growth-factor-beta-like signaling pathway can disrupt dorsal closure. Our findings show that although these pathways are linked they are not equivalent in function. Signaling by the Jun-amino-terminal-kinase cascade may be initiated by ...
Asymmetric cell divisions can be mediated by the preferential segregation of intrinsic cell fate ... more Asymmetric cell divisions can be mediated by the preferential segregation of intrinsic cell fate determinants into one of two sibling daughters. In dividing Drosophila neural progenitors the apical-basal orientation of the mitotic spindle, the basal cortical localization of the cell fate determinants Numb and/or Prospero as well as the coordination of these events are mediated by several proteins which include Bazooka (Baz), Inscuteable (Insc) and Partner of Inscuteable (Pins) which localize as an apical cortical complex starting at interphase. Here I will summarize data which suggest that the formation of this apical complex involves two distinct steps: (1) during the initiation of apical complex formation in interphase neuroblasts, there appears to be a hierarchical relation amongst these components where Baz recruits Insc and Baz/Insc in turn recruit Pins to the apical cortex/stalk; (2) while in delaminated mitotic neuroblasts the maintenance of the apical cortical localization of these proteins is dependent on the presence of all three components. Moreover, we show that the maintenance of this apical protein complex is essential for the correct execution of asymmetric division. Finally, the localization of the various asymmetrically localized proteins shows cell cycle dependence; however, the involvement of the cell cycle regulator in asymmetric cell divisions has not been previously shown. Here we present evidence from ongoing experiments which suggest a requirement for the key cell cycle regulator cdc2 in asymmetric cell divisions.
Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lact... more Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase from Bacillus stearothermophilus were made to try to change the substrate specificity from lactate to malate: Asp197----Asn, Thr246----Gly, and Gln102----Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln102----Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate (kcat) to Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1, equal to that of native lactate dehydrogenase for its natural substrate, pyruvate, and a maximum velocity (250 s-1), which is double that reported for a natural malate dehydrogenase from B. stearothermophilus.
Proceedings of the National Academy of Sciences, 1995
Nramp (natural resistance-associated macrophage protein) is a newly identified family of integral... more Nramp (natural resistance-associated macrophage protein) is a newly identified family of integral membrane proteins whose biochemical function is unknown. We report on the identification of Nramp homologs from the fly Drosophila melanogaster, the plant Oryza sativa, and the yeast Saccharomyces cerevisiae. Optimal alignment of protein sequences required insertion of very few gaps and revealed remarkable sequence identity of 28% (yeast), 40% (plant), and 55% (fly) with the mammalian proteins (46%, 58%, and 73% similarity), as well as a common predicted transmembrane topology. This family is defined by a highly conserved hydrophobic core encoding 10 transmembrane segments. Other features of this hydrophobic core include several invariant charged residues, helical periodicity of sequence conservation suggesting conserved and nonconserved faces for several transmembrane helices, a consensus transport signature on the intracytoplasmic face of the membrane, and structural determinants prev...
Asymmetric cell divisions can be mediated by the preferential segregation of cell-fate determinan... more Asymmetric cell divisions can be mediated by the preferential segregation of cell-fate determinants into one of two sibling daughters. In Drosophila neural progenitors, Inscuteable, Partner of Inscuteable and Bazooka localize as an apical cortical complex at interphase, which directs the apical-basal orientation of the mitotic spindle as well as the basal/cortical localization of the cell-fate determinants Numb and/or Prospero during mitosis. Although localization of these proteins shows dependence on the cell cycle, the involvement of cell-cycle components in asymmetric divisions has not been demonstrated. Here we show that neural progenitor asymmetric divisions require the cell-cycle regulator cdc2. By attenuating Drosophila cdc2 function without blocking mitosis, normally asymmetric progenitor divisions become defective, failing to correctly localize asymmetric components during mitosis and/or to resolve distinct sibling fates. cdc2 is not necessary for initiating apical complex formation during interphase; however, maintaining the asymmetric localization of the apical components during mitosis requires Cdc2/B-type cyclin complexes. Our findings link cdc2 with asymmetric divisions, and explain why the asymmetric localization of molecules like Inscuteable show cell-cycle dependence.
Cytoplasmic dynein is a multisubunit, microtubule-dependent motor enzyme that has been proposed t... more Cytoplasmic dynein is a multisubunit, microtubule-dependent motor enzyme that has been proposed to function in a variety of intracellular movements. As part of an effort to understand the evolution and the biological roles of cytoplasmic dynein, we have identified the first non-metazoan dynein light chain 1, SLC1, in the yeast Saccharomyces cerevisiae. The amino acid sequence of the SLC1 protein is similar to those of the human, Drosophila and Caenorhabditis cytoplasmic dynein light chains 1. The SLC1 gene lies adjacent to the YAP2 (= CAD1) transcription unit. The SLC1 coding sequence is split by two introns and its mRNA is detectable throughout the cell cycle. Tetrad analysis of heterozygotes harboring a TRP insertion in the SLC1 coding region indicate that SLC1 function is not essential for cell viability. Furthermore, we demonstrate that double mutants, defective for SLC1 and the kinesin-related CIN8 genes are non-lethal. The redundancy of SLC1 function in yeast contrasts with the cell death caused by loss-of-function mutations in the dynein light chain 1 gene in Drosophila melanogaster.
Asymmetric cell divisions generate cell fate diversity during both invertebrate and vertebrate de... more Asymmetric cell divisions generate cell fate diversity during both invertebrate and vertebrate development. Drosophila neural progenitors or neuroblasts (NBs) each divide asymmetrically to produce a larger neuroblast and a smaller ganglion mother cell (GMC). The asymmetric localisation of neural cell fate determinants and their adapter proteins to the neuroblast cortex during mitosis facilitates their preferential segregation to the GMC upon cytokinesis. In this study we report a novel role for the anaphase-promoting complex/cyclosome (APC/C) during this process. Attenuation of APC/C activity disrupts the asymmetric localisation of the adapter protein Miranda and its associated cargo proteins Staufen, Prospero and Brat, but not other components of the asymmetric division machinery. We demonstrate that Miranda is ubiquitylated via its C-terminal domain; removal of this domain disrupts Miranda localisation and replacement of this domain with a ubiquitin moiety restores normal asymmetr...
The neural precursor cells (sensory organ precursor cell (SOP)) of the external sense organs of D... more The neural precursor cells (sensory organ precursor cell (SOP)) of the external sense organs of Drosophila melanogaster arise from proneural clusters, which are defined through the expression pattern of proneural genes such as the genes of the achaete-scute complex (AS-C). The activities of these genes enable each cell within a cluster to become the SOP. A selection process mediated by
During Drosophila embryogenesis the two halves of the lateral epidermis migrate dorsally over a s... more During Drosophila embryogenesis the two halves of the lateral epidermis migrate dorsally over a surface of flattened cells, the amnioserosa, and meet at the dorsal midline in order to form the continuous sheet of the larval epidermis. During this process of epithelial migration, known as dorsal closure, signaling from a Jun-amino-terminal-kinase cascade causes the production of the secreted transforming-growth-factor-beta-like ligand, Decapentaplegic. Binding of Decapentaplegic to the putative transforming-growth-factor-beta-like receptors Thickveins and Punt activates a transforming-growth-factor-beta-like pathway that is also required for dorsal closure. Mutations in genes involved in either the Jun-amino-terminal-kinase cascade or the transforming-growth-factor-beta-like signaling pathway can disrupt dorsal closure. Our findings show that although these pathways are linked they are not equivalent in function. Signaling by the Jun-amino-terminal-kinase cascade may be initiated by ...
Asymmetric cell divisions can be mediated by the preferential segregation of intrinsic cell fate ... more Asymmetric cell divisions can be mediated by the preferential segregation of intrinsic cell fate determinants into one of two sibling daughters. In dividing Drosophila neural progenitors the apical-basal orientation of the mitotic spindle, the basal cortical localization of the cell fate determinants Numb and/or Prospero as well as the coordination of these events are mediated by several proteins which include Bazooka (Baz), Inscuteable (Insc) and Partner of Inscuteable (Pins) which localize as an apical cortical complex starting at interphase. Here I will summarize data which suggest that the formation of this apical complex involves two distinct steps: (1) during the initiation of apical complex formation in interphase neuroblasts, there appears to be a hierarchical relation amongst these components where Baz recruits Insc and Baz/Insc in turn recruit Pins to the apical cortex/stalk; (2) while in delaminated mitotic neuroblasts the maintenance of the apical cortical localization of these proteins is dependent on the presence of all three components. Moreover, we show that the maintenance of this apical protein complex is essential for the correct execution of asymmetric division. Finally, the localization of the various asymmetrically localized proteins shows cell cycle dependence; however, the involvement of the cell cycle regulator in asymmetric cell divisions has not been previously shown. Here we present evidence from ongoing experiments which suggest a requirement for the key cell cycle regulator cdc2 in asymmetric cell divisions.
Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lact... more Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase from Bacillus stearothermophilus were made to try to change the substrate specificity from lactate to malate: Asp197----Asn, Thr246----Gly, and Gln102----Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln102----Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate (kcat) to Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1, equal to that of native lactate dehydrogenase for its natural substrate, pyruvate, and a maximum velocity (250 s-1), which is double that reported for a natural malate dehydrogenase from B. stearothermophilus.
Proceedings of the National Academy of Sciences, 1995
Nramp (natural resistance-associated macrophage protein) is a newly identified family of integral... more Nramp (natural resistance-associated macrophage protein) is a newly identified family of integral membrane proteins whose biochemical function is unknown. We report on the identification of Nramp homologs from the fly Drosophila melanogaster, the plant Oryza sativa, and the yeast Saccharomyces cerevisiae. Optimal alignment of protein sequences required insertion of very few gaps and revealed remarkable sequence identity of 28% (yeast), 40% (plant), and 55% (fly) with the mammalian proteins (46%, 58%, and 73% similarity), as well as a common predicted transmembrane topology. This family is defined by a highly conserved hydrophobic core encoding 10 transmembrane segments. Other features of this hydrophobic core include several invariant charged residues, helical periodicity of sequence conservation suggesting conserved and nonconserved faces for several transmembrane helices, a consensus transport signature on the intracytoplasmic face of the membrane, and structural determinants prev...
Asymmetric cell divisions can be mediated by the preferential segregation of cell-fate determinan... more Asymmetric cell divisions can be mediated by the preferential segregation of cell-fate determinants into one of two sibling daughters. In Drosophila neural progenitors, Inscuteable, Partner of Inscuteable and Bazooka localize as an apical cortical complex at interphase, which directs the apical-basal orientation of the mitotic spindle as well as the basal/cortical localization of the cell-fate determinants Numb and/or Prospero during mitosis. Although localization of these proteins shows dependence on the cell cycle, the involvement of cell-cycle components in asymmetric divisions has not been demonstrated. Here we show that neural progenitor asymmetric divisions require the cell-cycle regulator cdc2. By attenuating Drosophila cdc2 function without blocking mitosis, normally asymmetric progenitor divisions become defective, failing to correctly localize asymmetric components during mitosis and/or to resolve distinct sibling fates. cdc2 is not necessary for initiating apical complex formation during interphase; however, maintaining the asymmetric localization of the apical components during mitosis requires Cdc2/B-type cyclin complexes. Our findings link cdc2 with asymmetric divisions, and explain why the asymmetric localization of molecules like Inscuteable show cell-cycle dependence.
Cytoplasmic dynein is a multisubunit, microtubule-dependent motor enzyme that has been proposed t... more Cytoplasmic dynein is a multisubunit, microtubule-dependent motor enzyme that has been proposed to function in a variety of intracellular movements. As part of an effort to understand the evolution and the biological roles of cytoplasmic dynein, we have identified the first non-metazoan dynein light chain 1, SLC1, in the yeast Saccharomyces cerevisiae. The amino acid sequence of the SLC1 protein is similar to those of the human, Drosophila and Caenorhabditis cytoplasmic dynein light chains 1. The SLC1 gene lies adjacent to the YAP2 (= CAD1) transcription unit. The SLC1 coding sequence is split by two introns and its mRNA is detectable throughout the cell cycle. Tetrad analysis of heterozygotes harboring a TRP insertion in the SLC1 coding region indicate that SLC1 function is not essential for cell viability. Furthermore, we demonstrate that double mutants, defective for SLC1 and the kinesin-related CIN8 genes are non-lethal. The redundancy of SLC1 function in yeast contrasts with the cell death caused by loss-of-function mutations in the dynein light chain 1 gene in Drosophila melanogaster.
Asymmetric cell divisions generate cell fate diversity during both invertebrate and vertebrate de... more Asymmetric cell divisions generate cell fate diversity during both invertebrate and vertebrate development. Drosophila neural progenitors or neuroblasts (NBs) each divide asymmetrically to produce a larger neuroblast and a smaller ganglion mother cell (GMC). The asymmetric localisation of neural cell fate determinants and their adapter proteins to the neuroblast cortex during mitosis facilitates their preferential segregation to the GMC upon cytokinesis. In this study we report a novel role for the anaphase-promoting complex/cyclosome (APC/C) during this process. Attenuation of APC/C activity disrupts the asymmetric localisation of the adapter protein Miranda and its associated cargo proteins Staufen, Prospero and Brat, but not other components of the asymmetric division machinery. We demonstrate that Miranda is ubiquitylated via its C-terminal domain; removal of this domain disrupts Miranda localisation and replacement of this domain with a ubiquitin moiety restores normal asymmetr...
The neural precursor cells (sensory organ precursor cell (SOP)) of the external sense organs of D... more The neural precursor cells (sensory organ precursor cell (SOP)) of the external sense organs of Drosophila melanogaster arise from proneural clusters, which are defined through the expression pattern of proneural genes such as the genes of the achaete-scute complex (AS-C). The activities of these genes enable each cell within a cluster to become the SOP. A selection process mediated by
Uploads