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1.
J Neurosci ; 21(24): 9529-40, 2001 Dec 15.
Article in English | MEDLINE | ID: mdl-11739564

ABSTRACT

Mutations in the potassium channel subunit KCNQ2 lead to benign familial neonatal convulsions, a dominantly inherited form of generalized epilepsy. In heterologous cells, KCNQ2 expression yields voltage-gated potassium channels that activate slowly (tau, approximately 0.1 sec) at subthreshold membrane potentials. KCNQ2 associates with KCNQ3, a homolog, to form heteromeric channels responsible for the M current (I(M)) in superior cervical ganglion (SCG) neurons. Muscarinic acetylcholine and peptidergic receptors inhibit SCG I(M), causing slow EPSPs and enhancing excitability. Here, we use KCNQ2N antibodies, directed against a conserved N-terminal portion of the KCNQ2 polypeptide, to localize KCNQ2-containing channels throughout mouse brain. We show that KCNQ2N immunoreactivity, although widespread, is particularly concentrated at key sites for control of rhythmic neuronal activity and synchronization. In the basal ganglia, we find KCNQ2N immunoreactivity on somata of dopaminergic and parvalbumin (PV)-positive (presumed GABAergic) cells of the substantia nigra, cholinergic large aspiny neurons of the striatum, and GABAergic and cholinergic neurons of the globus pallidus. In the septum, GABAergic, purinergic, and cholinergic neurons that contribute to the septohippocampal and septohabenular pathways exhibit somatic KCNQ2 labeling. In the thalamus, GABAergic nucleus reticularis neurons that regulate thalamocortical oscillations show strong labeling. In the hippocampus, many PV-positive and additional PV-negative interneurons exhibit strong somatic staining, but labeling of pyramidal and dentate granule somata is weak. There is strong neuropil staining in many regions. In some instances, notably the hippocampal mossy fibers, evidence indicates this neuropil staining is presynaptic.


Subject(s)
Biological Clocks/physiology , Brain/metabolism , Nerve Net/metabolism , Potassium Channels/metabolism , Protein Subunits , Animals , Antibodies/pharmacology , Antibody Specificity , Brain/cytology , Cells, Cultured , Conserved Sequence/physiology , Epilepsy, Benign Neonatal/genetics , Humans , Immunohistochemistry , KCNQ2 Potassium Channel , KCNQ3 Potassium Channel , Kidney/cytology , Kidney/metabolism , Male , Mice , Mice, Inbred C57BL , Nerve Net/cytology , Organ Specificity , Periodicity , Potassium Channel Blockers , Potassium Channels/genetics , Potassium Channels, Voltage-Gated , Transfection
2.
Nature ; 414(6864): 634-8, 2001 Dec 06.
Article in English | MEDLINE | ID: mdl-11740559

ABSTRACT

Establishing cellular polarity is critical for tissue organization and function. Initially discovered in the landmark genetic screen for Drosophila developmental mutants, bazooka, crumbs, shotgun and stardust mutants exhibit severe disruption in apicobasal polarity in embryonic epithelia, resulting in multilayered epithelia, tissue disintegration, and defects in cuticle formation. Here we report that stardust encodes single PDZ domain MAGUK (membrane-associated guanylate kinase) proteins that are expressed in all primary embryonic epithelia from the onset of gastrulation. Stardust colocalizes with Crumbs at the apicolateral boundary, although their expression patterns in sensory organs differ. Stardust binds to the carboxy terminus of Crumbs in vitro, and Stardust and Crumbs are mutually dependent in their stability, localization and function in controlling the apicobasal polarity of epithelial cells. However, for the subset of ectodermal cells that delaminate and form neuroblasts, their polarity requires the function of Bazooka, but not of Stardust or Crumbs.


Subject(s)
Cell Polarity , Drosophila Proteins/physiology , Epithelial Cells/cytology , Intracellular Signaling Peptides and Proteins , Membrane Proteins/physiology , Membrane Transport Proteins , Neurons/cytology , Nucleoside-Phosphate Kinase/physiology , Adherens Junctions/physiology , Amino Acid Sequence , Animals , Carrier Proteins/metabolism , Cloning, Molecular , DNA, Complementary , Drosophila/cytology , Drosophila/embryology , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Guanylate Kinases , Membrane Proteins/metabolism , Molecular Sequence Data , Mutation , Nucleoside-Phosphate Kinase/genetics , Nucleoside-Phosphate Kinase/metabolism , Protein Binding , Protein Kinase C/metabolism , Proteins/metabolism
3.
Proc Natl Acad Sci U S A ; 98(25): 14469-74, 2001 Dec 04.
Article in English | MEDLINE | ID: mdl-11734647

ABSTRACT

Asymmetric divisions with two different division orientations follow different polarity cues for the asymmetric segregation of determinants in the sensory organ precursor (SOP) lineage. The first asymmetric division depends on frizzled function and has the mitotic spindle of the pI cell in the epithelium oriented along the anterior-posterior axis, giving rise to pIIa and pIIb, which divide in different orientations. Only the pIIb division resembles neuroblast division in daughter-size asymmetry, spindle orientation along the apical-basal axis, basal Numb localization, and requirement for inscuteable function. Because the PDZ domain protein Bazooka is required for spindle orientation and basal localization of Numb in neuroblasts, we wondered whether Bazooka plays a similar role in the pIIb in the SOP lineage. Surprisingly, Bazooka controls asymmetric localization of the Numb-anchoring protein Pon, but not spindle orientation, in pI and all subsequent divisions. Bazooka also regulates cell proliferation in the SOP lineage; loss of bazooka function results in supernumerary cell divisions and apoptotic cell death.


Subject(s)
Carrier Proteins/physiology , Drosophila Proteins/physiology , Drosophila/cytology , Intracellular Signaling Peptides and Proteins , Animals , Apoptosis/physiology , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Division/physiology , Cell Polarity/physiology , Drosophila/genetics , Drosophila/physiology , Drosophila Proteins/genetics , Genes, Insect , Mutation , Sense Organs/cytology , Stem Cells/cytology
4.
Proc Natl Acad Sci U S A ; 98(25): 14475-80, 2001 Dec 04.
Article in English | MEDLINE | ID: mdl-11734648

ABSTRACT

The par genes, identified by their role in the establishment of anterior-posterior polarity in the Caenorhabditis elegans zygote, subsequently have been shown to regulate cellular polarity in diverse cell types by means of an evolutionarily conserved protein complex including PAR-3, PAR-6, and atypical protein kinase C (aPKC). The Drosophila homologs of par-1, par-3 (bazooka, baz), par-6 (DmPar-6), and pkc-3 (Drosophila aPKC, DaPKC) each are known to play conserved roles in the generation of cell polarity in the germ line as well as in epithelial and neural precursor cells within the embryo. In light of this functional conservation, we examined the potential role of baz and DaPKC in the regulation of oocyte polarity. Our analyses reveal germ-line autonomous roles for baz and DaPKC in the establishment of initial anterior-posterior polarity within germ-line cysts and maintenance of oocyte cell fate. Germ-line clonal analyses indicate both proteins are essential for two key aspects of oocyte determination: the posterior translocation of oocyte specification factors and the posterior establishment of the microtubule organizing center within the presumptive oocyte. We demonstrate BAZ and DaPKC colocalize to belt-like structures between germarial cyst cells. However, in contrast to their regulatory relationship in the Drosophila and C. elegans embryos, these proteins are not mutually dependent for their germ-line localization, nor is either protein specifically required for PAR-1 localization to the fusome. Therefore, whereas BAZ, DaPKC, and PAR-1 are functionally conserved in establishing oocyte polarity, the regulatory relationships among these genes are not well conserved, indicating these molecules function differently in different cellular contexts.


Subject(s)
Caenorhabditis elegans Proteins , Carrier Proteins/physiology , Drosophila Proteins/physiology , Drosophila/cytology , Intracellular Signaling Peptides and Proteins , Oocytes/cytology , Protein Kinase C/physiology , Trans-Activators , Animals , Armadillo Domain Proteins , Cadherins/metabolism , Carrier Proteins/genetics , Cell Differentiation/physiology , Cell Polarity/physiology , Drosophila/genetics , Drosophila/physiology , Drosophila Proteins/genetics , Female , Genes, Insect , Immunohistochemistry , Insect Proteins/metabolism , Oogenesis/genetics , Oogenesis/physiology , Ovary/cytology , Protein Kinase C/genetics , Protein Serine-Threonine Kinases/metabolism , Transcription Factors
5.
Dev Cell ; 1(5): 667-77, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11709187

ABSTRACT

Morphological complexity of neurons contributes to their functional complexity. How neurons generate different dendritic patterns is not known. We identified the sequoia mutant from a previous screen for dendrite mutants. Here we report that Sequoia is a pan-neural nuclear protein containing two putative zinc fingers homologous to the DNA binding domain of Tramtrack. sequoia mutants affect the cell fate decision of a small subset of neurons but have global effects on axon and dendrite morphologies of most and possibly all neurons. In support of sequoia as a specific regulator of neuronal morphogenesis, microarray experiments indicate that sequoia may regulate downstream genes that are important for executing neurite development rather than altering a variety of molecules that specify cell fates.


Subject(s)
Axons/metabolism , DNA-Binding Proteins/metabolism , Dendrites/metabolism , Drosophila Proteins , Drosophila/embryology , Nerve Tissue Proteins/metabolism , Nervous System/cytology , Nervous System/embryology , Repressor Proteins/chemistry , Zinc Fingers , Amino Acid Sequence , Animals , Cell Differentiation , Cell Division , Cell Lineage , Cell Nucleus/metabolism , Cell Size , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Drosophila/cytology , Drosophila/genetics , Gene Expression Regulation, Developmental , In Situ Hybridization , Molecular Sequence Data , Mutation/genetics , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Amino Acid
6.
Proc Natl Acad Sci U S A ; 98(25): 14649-54, 2001 Dec 04.
Article in English | MEDLINE | ID: mdl-11724956

ABSTRACT

Many G protein-coupled receptors (GPCRs) have recently been shown to dimerize, and it was suggested that dimerization may be a prerequisite for G protein coupling. gamma-aminobutyric acid type B (GABA(B)) receptors (GPCRs for GABA, a major inhibitory neurotransmitter in the brain) are obligate heterodimers of homologous GB1 and GB2 subunits, neither of which is functional on its own. This feature of GABA(B) receptors allowed us to examine which of the eight intracellular segments of the heterodimeric receptor were important for G protein activation. Replacing any of the three intracellular loops of GB2 with their GB1 counterparts resulted in nonfunctional receptors. The deletion of the complete GB2 C terminus significantly attenuated the receptor function; however, the proximal 36 residues were sufficient for reconstitution of wild type-like receptor activity. In contrast, the GB1 C terminus could be deleted and GB1 intracellular loops replaced with their GB2 or mGluR1 equivalents without affecting the receptor function. In addition, a large portion of the GB1 i2 loop could be replaced with a random coil peptide without any functional consequences. Thus, GB2 intracellular segments are solely responsible for specific coupling of GABA(B) receptors to their physiologic effectors, G(i) and G protein-activated K(+) channels. These findings strongly support a model in which a single GPCR monomer is sufficient for all of the specific G protein contacts.


Subject(s)
GTP-Binding Proteins/metabolism , Receptors, GABA-B/chemistry , Receptors, GABA-B/metabolism , Amino Acid Sequence , Animals , Female , In Vitro Techniques , Models, Neurological , Molecular Sequence Data , Mutation , Oocytes/metabolism , Protein Structure, Tertiary , Protein Subunits , Receptors, GABA-B/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Xenopus
7.
Proc Natl Acad Sci U S A ; 98(25): 14643-8, 2001 Dec 04.
Article in English | MEDLINE | ID: mdl-11724957

ABSTRACT

gamma-aminobutyric acid type B (GABA(B)) receptors, G protein-coupled receptors (GPCRs) for GABA, are obligate heterodimers of two homologous subunits, GB1 and GB2. Typical for family C GPCRs, the N termini of both GB1 and GB2 contain a domain with homology to bacterial periplasmic amino acid-binding proteins (PBPs), but only the GB1 PBP-like domain binds GABA. We found that both GB1 and GB2 extracellular N termini are required for normal coupling of GABA(B) receptors to their physiological effectors, G(i) and G protein-activated K(+) channels (GIRKs). Receptors with two GB2 N termini did not respond to GABA, whereas receptors with two GB1 N termini showed increased basal activity and responded to GABA with inhibition, rather than activation, of GIRK channels. This GABA-induced GIRK current inhibition depended on GABA binding to the chimeric GB(1/2) subunit (the GB1 N-terminal domain attached to the heptahelical domain of GB2), rather than the wild-type GB1 subunit. Interestingly, receptors with reciprocal exchange of N-terminal domains between the subunits were functionally indistinguishable from wild-type receptors. We also found that peptide linkers between GB1 and GB2 PBP-like domains and respective heptahelical domains could be altered without affecting receptor function. This finding suggests that other contacts between the PBP-like and heptahelical domains underlie ligand-induced signal transduction, a finding likely to be relevant for all family C GPCRs.


Subject(s)
Potassium Channels, Inwardly Rectifying , Receptors, GABA-B/metabolism , Amino Acid Sequence , Animals , Binding Sites/genetics , Female , G Protein-Coupled Inwardly-Rectifying Potassium Channels , In Vitro Techniques , Ligands , Models, Neurological , Molecular Sequence Data , Mutation , Oocytes/metabolism , Potassium Channels , Protein Structure, Tertiary , Protein Subunits , Receptors, GABA-B/chemistry , Receptors, GABA-B/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Xenopus
9.
Genes Dev ; 15(20): 2627-41, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11641269
10.
Proc Natl Acad Sci U S A ; 98(20): 11016-23, 2001 Sep 25.
Article in English | MEDLINE | ID: mdl-11572962

ABSTRACT

Neural signaling is based on the regulated timing and extent of channel opening; therefore, it is important to understand how ion channels open and close in response to neurotransmitters and intracellular messengers. Here, we examine this question for potassium channels, an extraordinarily diverse group of ion channels. Voltage-gated potassium (Kv) channels control action-potential waveforms and neuronal firing patterns by opening and closing in response to membrane-potential changes. These effects can be strongly modulated by cytoplasmic factors such as kinases, phosphatases, and small GTPases. A Kv alpha subunit contains six transmembrane segments, including an intrinsic voltage sensor. In contrast, inwardly rectifying potassium (Kir) channels have just two transmembrane segments in each of its four pore-lining alpha subunits. A variety of intracellular second messengers mediate transmitter and metabolic regulation of Kir channels. For example, Kir3 (GIRK) channels open on binding to the G protein betagamma subunits, thereby mediating slow inhibitory postsynaptic potentials in the brain. Our structure-based functional analysis on the cytoplasmic N-terminal tetramerization domain T1 of the voltage-gated channel, Kv1.2, uncovered a new function for this domain, modulation of voltage gating, and suggested a possible means of communication between second messenger pathways and Kv channels. A yeast screen for active Kir3.2 channels subjected to random mutagenesis has identified residues in the transmembrane segments that are crucial for controlling the opening of Kir3.2 channels. The identification of structural elements involved in potassium channel gating in these systems highlights principles that may be important in the regulation of other types of channels.


Subject(s)
Cell Membrane/physiology , Potassium Channels/physiology , Amino Acid Sequence , Animals , Ion Channel Gating , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Potassium Channels/chemistry , Protein Conformation , Signal Transduction/physiology
11.
Proc Natl Acad Sci U S A ; 98(17): 9907-12, 2001 Aug 14.
Article in English | MEDLINE | ID: mdl-11481456

ABSTRACT

Neurogenic genes in the Notch receptor-mediated signaling pathway play important roles in neuronal cell fate specification as well as neuronal differentiation. The Drosophila neuralized gene is one of the neurogenic genes. We have cloned a mouse homolog of Drosophila neuralized, m-neu1, and found that the m-neu1 transcript is expressed in differentiated neurons. Mice deficient for m-neu1 are viable and morphologically normal, but exhibit specific defects in olfactory discrimination and hypersensitivity to ethanol. These findings reveal an essential role of m-neu1 in ensuring proper processing of certain information in the adult brain.


Subject(s)
Discrimination, Psychological , Drosophila Proteins , Ethanol/toxicity , Ligases , Nerve Tissue Proteins/physiology , Olfaction Disorders/genetics , Smell/genetics , Ubiquitin-Protein Ligases , Amino Acid Sequence , Animals , Ataxia/chemically induced , Ataxia/genetics , Avoidance Learning , Blotting, Northern , Brain Chemistry , DNA, Complementary/genetics , Drosophila melanogaster/genetics , Drug Resistance/genetics , Gene Library , Genotype , Hemiterpenes , Insect Proteins/genetics , Male , Maze Learning , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Neurons/metabolism , Odorants , Pentanoic Acids , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity
12.
Nat Cell Biol ; 3(7): 628-36, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11433294

ABSTRACT

Wnt signalling regulates beta-catenin-dependent developmental processes through the Dishevelled protein (Dsh). Dsh regulates two distinct pathways, one mediated by beta-catenin and the other by Jun kinase (JNK). We have purified a Dsh-associated kinase from Drosophila that encodes a homologue of Caenorhabditis elegans PAR-1, a known determinant of polarity during asymmetric cell divisions. Treating cells with Wnt increases endogenous PAR-1 activity coincident with Dsh phosphorylation. PAR-1 potentiates Wnt activation of the beta-catenin pathway but blocks the JNK pathway. Suppressing endogenous PAR-1 function inhibits Wnt signalling through beta-catenin in mammalian cells, and Xenopus and Drosophila embryos. PAR-1 seems to be a positive regulator of the beta-catenin pathway and an inhibitor of the JNK pathway. These findings show that PAR-1, a regulator of polarity, is also a modulator of Wnt-beta-catenin signalling, indicating a link between two important developmental pathways.


Subject(s)
Caenorhabditis elegans Proteins , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins/physiology , Trans-Activators , Zebrafish Proteins , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cytoskeletal Proteins/drug effects , Dishevelled Proteins , Drosophila/embryology , Drosophila Proteins , Humans , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/drug effects , Mitogens/physiology , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/pharmacology , Proto-Oncogene Proteins/drug effects , Proto-Oncogene Proteins/pharmacology , Sequence Alignment , Signal Transduction/drug effects , Transcriptional Activation/drug effects , Tumor Cells, Cultured , Wnt Proteins , Xenopus , Xenopus Proteins , beta Catenin
13.
Neuron ; 29(3): 657-67, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11301025

ABSTRACT

GIRK2 is a major contributor to G protein-activated inward rectifier potassium channels in the mammalian brain. How GIRK channels open upon contact with Gbetagamma remains unknown. Using a yeast genetic screen to select constitutively active mutants from a randomly mutagenized GIRK2 library, we identified five gating mutations at four residues in the transmembrane domain. Further mutagenesis indicates that GIRK channel opening involves a rotation of the transmembrane segments, bringing one of these residues (V188) to a pore-lining position in the open conformation. Combined with double-mutant studies, these findings suggest that GIRK channels gate by moving from the open conformation inferred from our yeast study of Kir2.1 to a closed conformation perhaps resembling the known KcsA structure.


Subject(s)
GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , Heterotrimeric GTP-Binding Proteins/metabolism , Mutagenesis , Potassium Channels, Inwardly Rectifying , Potassium Channels/genetics , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Animals , DNA Mutational Analysis , Electric Conductivity , Female , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Gene Expression , Ion Channel Gating/genetics , Kinetics , Models, Molecular , Molecular Sequence Data , Oocytes/metabolism , Potassium Channels/chemistry , Potassium Channels/physiology , Protein Conformation , Sequence Alignment , Structure-Activity Relationship , Xenopus
14.
Curr Biol ; 11(2): 75-87, 2001 Jan 23.
Article in English | MEDLINE | ID: mdl-11231123

ABSTRACT

BACKGROUND: Drosophila oocyte determination involves a complex process by which a single cell within an interconnected cyst of 16 germline cells differentiates into an oocyte. This process requires the asymmetric accumulation of both specific messenger RNAs and proteins within the future oocyte as well as the proper organization of the microtubule cytoskeleton, which together with the fusome provides polarity within the developing germline cyst. RESULTS: In addition to its previously described late oogenic role in the establishment of anterior-posterior polarity and subsequent embryonic axis formation, the Drosophila par-1 gene is required very early in the germline for establishing cyst polarity and for oocyte specification. Germline clonal analyses, for which we used a protein null mutation, reveal that Drosophila par-1 (par-1) is required for the asymmetric accumulation of oocyte-specific factors as well as the proper organization of the microtubule cytoskeleton. Similarly, somatic clonal analyses indicate that par-1 is required for microtubule stabilization in follicle cells. The PAR-1 protein is localized to the fusome and ring canals within the developing germline cyst in direct contact with microtubules. Likewise, in the follicular epithelium, PAR-1 colocalizes with microtubules along the basolateral membrane. However, in either case PAR-1 localization is independent of microtubules. CONCLUSIONS: The Drosophila par-1 gene plays at least two essential roles during oogenesis; it is required early in the germline for organization of the microtubule cytoskeleton and subsequent oocyte determination, and it has a second, previously described role late in oogenesis in axis formation. In both cases, par-1 appears to exert its effects through the regulation of microtubule dynamics and/or stability, and this finding is consistent with the defined role of the mammalian PAR-1 homologs.


Subject(s)
Caenorhabditis elegans Proteins , Cell Differentiation/genetics , Drosophila/genetics , Microtubules/ultrastructure , Oocytes/cytology , Protein Serine-Threonine Kinases/genetics , Animals , Drosophila/cytology , Immunohistochemistry , In Situ Hybridization , Mutation
15.
Proc Natl Acad Sci U S A ; 98(5): 2431-6, 2001 Feb 27.
Article in English | MEDLINE | ID: mdl-11226256

ABSTRACT

To improve the accuracy of predicting membrane protein sorting signals, we developed a general methodology for defining trafficking signal consensus sequences in the environment of the living cell. Our approach uses retroviral gene transfer to create combinatorial expression libraries of trafficking signal variants in mammalian cells, flow cytometry to sort cells based on trafficking phenotype, and quantitative trafficking assays to measure the efficacy of individual signals. Using this strategy to analyze arginine- and lysine-based endoplasmic reticulum localization signals, we demonstrate that small changes in the local sequence context dramatically alter signal strength, generating a broad spectrum of trafficking phenotypes. Finally, using sequences from our screen, we found that the potency of di-lysine, but not di-arginine, mediated endoplasmic reticulum localization was correlated with the strength of interaction with alpha-COP.


Subject(s)
Combinatorial Chemistry Techniques , Endoplasmic Reticulum/metabolism , Signal Transduction , Amino Acid Sequence , Animals , Flow Cytometry , Fluorescent Antibody Technique , Genes, Reporter , Golgi Apparatus/metabolism , Molecular Sequence Data , Two-Hybrid System Techniques
16.
Science ; 291(5502): 316-9, 2001 Jan 12.
Article in English | MEDLINE | ID: mdl-11209084

ABSTRACT

Little is known about the identity of endoplasmic reticulum (ER) export signals and how they are used to regulate the number of proteins on the cell surface. Here, we describe two ER export signals that profoundly altered the steady-state distribution of potassium channels and were required for channel localization to the plasma membrane. When transferred to other potassium channels or a G protein-coupled receptor, these ER export signals increased the number of functional proteins on the cell surface. Thus, ER export of membrane proteins is not necessarily limited by folding or assembly, but may be under the control of specific export signals.


Subject(s)
Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Potassium Channels, Inwardly Rectifying , Potassium Channels, Voltage-Gated , Potassium Channels/chemistry , Potassium Channels/metabolism , Protein Sorting Signals , 3T3 Cells , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution , Animals , COS Cells , Cell Line , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Glycosylation , Golgi Apparatus/metabolism , Kv1.2 Potassium Channel , Mice , Molecular Sequence Data , Oocytes , Potassium Channels/genetics , Protein Folding , Protein Transport , Receptors, GABA-B/chemistry , Receptors, GABA-B/metabolism , Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Retinoid X Receptors , Transcription Factors/chemistry , Transcription Factors/metabolism , Xenopus
17.
Nature ; 409(6819): 522-5, 2001 Jan 25.
Article in English | MEDLINE | ID: mdl-11206549

ABSTRACT

Asymmetric division is a fundamental mechanism for generating cellular diversity. In the central nervous system of Drosophila, neural progenitor cells called neuroblasts undergo asymmetric division along the apical-basal cellular axis. Neuroblasts originate from neuroepithelial cells, which are polarized along the apical-basal axis and divide symmetrically along the planar axis. The asymmetry of neuroblasts might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. Here we show that by disrupting adherens junctions we can convert the symmetric epithelial division into asymmetric division. We further confirm that the adenomatous polyposis coli (APC) tumour suppressor protein is recruited to adherens junctions, and demonstrate that both APC and microtubule-associated EB1 homologues are required for the symmetric epithelial division along the planar axis. Our results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions.


Subject(s)
Adherens Junctions/physiology , Cell Division/physiology , Drosophila Proteins , Neurons/cytology , Adenomatous Polyposis Coli Protein , Animals , Animals, Genetically Modified , Body Patterning/physiology , Carrier Proteins/physiology , Cell Differentiation , Cell Polarity , Cytoskeletal Proteins/physiology , Drosophila , Epithelial Cells/cytology , Humans , Spindle Apparatus/physiology
18.
Nat Cell Biol ; 3(1): 58-67, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11146627

ABSTRACT

Asymmetric partitioning of cell-fate determinants during development requires coordinating the positioning of these determinants with orientation of the mitotic spindle. In the Drosophila peripheral nervous system, sensory organ progenitor cells (SOPs) undergo several rounds of division to produce five cells that give rise to a complete sensory organ. Here we have observed the asymmetric divisions that give rise to these cells in the developing pupae using green fluorescent protein fusion proteins. We find that spindle orientation and determinant localization are tightly coordinated at each division. Furthermore, we find that two types of asymmetric divisions exist within the sensory organ precursor cell lineage: the anterior-posterior pI cell-type division, where the spindle remains symmetric throughout mitosis, and the strikingly neuroblast-like apical-basal division of the pIIb cell, where the spindle exhibits a strong asymmetry at anaphase. In both these divisions, the spindle reorientates to position itself perpendicular to the region of the cortex containing the determinant. On the basis of these observations, we propose that two distinct mechanisms for controlling asymmetric cell divisions occur within the same lineage in the developing peripheral nervous system in Drosophila.


Subject(s)
Cell Division/genetics , Cell Lineage/genetics , Drosophila Proteins , Drosophila/growth & development , Neurons, Afferent/metabolism , Sense Organs/growth & development , Stem Cells/metabolism , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Polarity/genetics , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Drosophila/cytology , Drosophila/metabolism , Frizzled Receptors , Gene Expression Regulation, Developmental , Green Fluorescent Proteins , Indicators and Reagents/analysis , Interphase/genetics , Juvenile Hormones/genetics , Juvenile Hormones/metabolism , Luminescent Proteins/analysis , Membrane Proteins/genetics , Membrane Proteins/metabolism , Neurons, Afferent/cytology , Neuropeptides , Pupa/cytology , Pupa/growth & development , Pupa/metabolism , Receptors, G-Protein-Coupled , Sense Organs/cytology , Sense Organs/metabolism , Spindle Apparatus/genetics , Spindle Apparatus/metabolism , Stem Cells/cytology
19.
Proc Natl Acad Sci U S A ; 97(25): 13726-31, 2000 Dec 05.
Article in English | MEDLINE | ID: mdl-11095759

ABSTRACT

Aging is a universal but poorly understood biological process. Free radicals accumulate with age and have been proposed to be a major cause of aging. We measured genome-wide changes in transcript levels as a function of age in Drosophila melanogaster and compared these changes with those caused by paraquat, a free-radical generator. A number of genes exhibited changes in transcript levels with both age and paraquat treatment. We also found genes whose transcript levels changed with age but not with paraquat treatment. This study suggests that free radicals play an important role in regulating transcript levels in aging but that they are not the only factors. This genome-wide survey also identifies candidates for molecular markers of aging.


Subject(s)
Aging/genetics , Drosophila melanogaster/physiology , Genome , Oxidative Stress/genetics , Animals , Drosophila melanogaster/genetics , Gene Expression Regulation/physiology , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproduction/genetics
20.
Neuron ; 28(1): 91-101, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11086986

ABSTRACT

Neurons elaborate dendrites with stereotypic branching patterns, thereby defining their receptive fields. These branching patterns may arise from properties intrinsic to the neurons or competition between neighboring neurons. Genetic and laser ablation studies reported here reveal that different multiple dendritic neurons in the same dorsal cluster in the Drosophila embryonic PNS do not compete with one another for dendritic fields. In contrast, when dendrites from homologous neurons in the two hemisegments meet at the dorsal midline in larval stages, they appear to repel each other. The formation of normal dendritic fields and the competition between dendrites of homologous neurons require the proper expression level of Flamingo, a G protein-coupled receptor-like protein, in embryonic neurons. Whereas Flamingo functions downstream of Frizzled in specifying planar polarity, Flamingo-dependent dendritic outgrowth is independent of Frizzled.


Subject(s)
Cadherins/metabolism , Dendrites/metabolism , Drosophila Proteins , Drosophila/embryology , Embryo, Nonmammalian/innervation , Neurons/metabolism , Animals , Cadherins/genetics , Dendrites/ultrastructure , Embryo, Nonmammalian/cytology , Frizzled Receptors , Larva/cytology , Membrane Proteins/genetics , Mutation , Neurons/ultrastructure , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled , Signal Transduction/genetics
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