RESUMEN
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.
Asunto(s)
Polaridad Celular , Proteínas de Drosophila/fisiología , Células Epiteliales/citología , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana , Neuronas/citología , Nucleósido-Fosfato Quinasa/fisiología , Uniones Adherentes/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Clonación Molecular , ADN Complementario , Drosophila/citología , Drosophila/embriología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Guanilato-Quinasas , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Nucleósido-Fosfato Quinasa/genética , Nucleósido-Fosfato Quinasa/metabolismo , Unión Proteica , Proteína Quinasa C/metabolismo , Proteínas/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de Drosophila/fisiología , Drosophila/citología , Péptidos y Proteínas de Señalización Intracelular , Animales , Apoptosis/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , División Celular/fisiología , Polaridad Celular/fisiología , Drosophila/genética , Drosophila/fisiología , Proteínas de Drosophila/genética , Genes de Insecto , Mutación , Órganos de los Sentidos/citología , Células Madre/citologíaRESUMEN
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.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas Portadoras/fisiología , Proteínas de Drosophila/fisiología , Drosophila/citología , Péptidos y Proteínas de Señalización Intracelular , Oocitos/citología , Proteína Quinasa C/fisiología , Transactivadores , Animales , Proteínas del Dominio Armadillo , Cadherinas/metabolismo , Proteínas Portadoras/genética , Diferenciación Celular/fisiología , Polaridad Celular/fisiología , Drosophila/genética , Drosophila/fisiología , Proteínas de Drosophila/genética , Femenino , Genes de Insecto , Inmunohistoquímica , Proteínas de Insectos/metabolismo , Oogénesis/genética , Oogénesis/fisiología , Ovario/citología , Proteína Quinasa C/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de TranscripciónRESUMEN
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.
Asunto(s)
Relojes Biológicos/fisiología , Encéfalo/metabolismo , Red Nerviosa/metabolismo , Canales de Potasio/metabolismo , Subunidades de Proteína , Animales , Anticuerpos/farmacología , Especificidad de Anticuerpos , Encéfalo/citología , Células Cultivadas , Secuencia Conservada/fisiología , Epilepsia Benigna Neonatal/genética , Humanos , Inmunohistoquímica , Canal de Potasio KCNQ2 , Canal de Potasio KCNQ3 , Riñón/citología , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Red Nerviosa/citología , Especificidad de Órganos , Periodicidad , Bloqueadores de los Canales de Potasio , Canales de Potasio/genética , Canales de Potasio con Entrada de Voltaje , TransfecciónRESUMEN
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.
Asunto(s)
Axones/metabolismo , Proteínas de Unión al ADN/metabolismo , Dendritas/metabolismo , Proteínas de Drosophila , Drosophila/embriología , Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso/citología , Sistema Nervioso/embriología , Proteínas Represoras/química , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Diferenciación Celular , División Celular , Linaje de la Célula , Núcleo Celular/metabolismo , Tamaño de la Célula , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Drosophila/citología , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Datos de Secuencia Molecular , Mutación/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores de GABA-B/química , Receptores de GABA-B/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Técnicas In Vitro , Modelos Neurológicos , Datos de Secuencia Molecular , Mutación , Oocitos/metabolismo , Estructura Terciaria de Proteína , Subunidades de Proteína , Receptores de GABA-B/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , XenopusRESUMEN
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.
Asunto(s)
Canales de Potasio de Rectificación Interna , Receptores de GABA-B/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Técnicas In Vitro , Ligandos , Modelos Neurológicos , Datos de Secuencia Molecular , Mutación , Oocitos/metabolismo , Canales de Potasio , Estructura Terciaria de Proteína , Subunidades de Proteína , Receptores de GABA-B/química , Receptores de GABA-B/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , XenopusAsunto(s)
División Celular/fisiología , Drosophila melanogaster/fisiología , Neuronas/fisiología , Animales , Linaje de la Célula , Polaridad Celular , Drosophila melanogaster/citología , Sistema Nervioso/citología , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Transducción de Señal/fisiología , Huso Acromático/metabolismoRESUMEN
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.
Asunto(s)
Membrana Celular/fisiología , Canales de Potasio/fisiología , Secuencia de Aminoácidos , Animales , Activación del Canal Iónico , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Canales de Potasio/química , Conformación Proteica , Transducción de Señal/fisiologíaRESUMEN
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.
Asunto(s)
Discriminación en Psicología , Proteínas de Drosophila , Etanol/toxicidad , Ligasas , Proteínas del Tejido Nervioso/fisiología , Trastornos del Olfato/genética , Olfato/genética , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Animales , Ataxia/inducido químicamente , Ataxia/genética , Reacción de Prevención , Northern Blotting , Química Encefálica , ADN Complementario/genética , Drosophila melanogaster/genética , Resistencia a Medicamentos/genética , Biblioteca de Genes , Genotipo , Hemiterpenos , Proteínas de Insectos/genética , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Odorantes , Ácidos Pentanoicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Proteínas de Caenorhabditis elegans , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transactivadores , Proteínas de Pez Cebra , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Proteínas del Citoesqueleto/efectos de los fármacos , Proteínas Dishevelled , Drosophila/embriología , Proteínas de Drosophila , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Mitógenos/fisiología , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/farmacología , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/farmacología , Alineación de Secuencia , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Células Tumorales Cultivadas , Proteínas Wnt , Xenopus , Proteínas de Xenopus , beta CateninaRESUMEN
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.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Mutagénesis , Canales de Potasio de Rectificación Interna , Canales de Potasio/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Análisis Mutacional de ADN , Conductividad Eléctrica , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Expresión Génica , Activación del Canal Iónico/genética , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Oocitos/metabolismo , Canales de Potasio/química , Canales de Potasio/fisiología , Conformación Proteica , Alineación de Secuencia , Relación Estructura-Actividad , XenopusRESUMEN
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.
Asunto(s)
Proteínas de Caenorhabditis elegans , Diferenciación Celular/genética , Drosophila/genética , Microtúbulos/ultraestructura , Oocitos/citología , Proteínas Serina-Treonina Quinasas/genética , Animales , Drosophila/citología , Inmunohistoquímica , Hibridación in Situ , MutaciónRESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/química , Canales de Potasio/metabolismo , Señales de Clasificación de Proteína , Células 3T3 , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células COS , Línea Celular , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Glicosilación , Aparato de Golgi/metabolismo , Canal de Potasio Kv.1.2 , Ratones , Datos de Secuencia Molecular , Oocitos , Canales de Potasio/genética , Pliegue de Proteína , Transporte de Proteínas , Receptores de GABA-B/química , Receptores de GABA-B/metabolismo , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Receptores X Retinoide , Factores de Transcripción/química , Factores de Transcripción/metabolismo , XenopusRESUMEN
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.
Asunto(s)
Uniones Adherentes/fisiología , División Celular/fisiología , Proteínas de Drosophila , Neuronas/citología , Proteína de la Poliposis Adenomatosa del Colon , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/fisiología , Proteínas Portadoras/fisiología , Diferenciación Celular , Polaridad Celular , Proteínas del Citoesqueleto/fisiología , Drosophila , Células Epiteliales/citología , Humanos , Huso Acromático/fisiologíaRESUMEN
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.
Asunto(s)
Técnicas Químicas Combinatorias , Retículo Endoplásmico/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Genes Reporteros , Aparato de Golgi/metabolismo , Datos de Secuencia Molecular , Técnicas del Sistema de Dos HíbridosRESUMEN
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.
Asunto(s)
División Celular/genética , Linaje de la Célula/genética , Proteínas de Drosophila , Drosophila/crecimiento & desarrollo , Neuronas Aferentes/metabolismo , Órganos de los Sentidos/crecimiento & desarrollo , Células Madre/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Polaridad Celular/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Drosophila/citología , Drosophila/metabolismo , Receptores Frizzled , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes , Indicadores y Reactivos/análisis , Interfase/genética , Hormonas Juveniles/genética , Hormonas Juveniles/metabolismo , Proteínas Luminiscentes/análisis , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neuronas Aferentes/citología , Neuropéptidos , Pupa/citología , Pupa/crecimiento & desarrollo , Pupa/metabolismo , Receptores Acoplados a Proteínas G , Órganos de los Sentidos/citología , Órganos de los Sentidos/metabolismo , Huso Acromático/genética , Huso Acromático/metabolismo , Células Madre/citologíaRESUMEN
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.
Asunto(s)
Envejecimiento/genética , Drosophila melanogaster/fisiología , Genoma , Estrés Oxidativo/genética , Animales , Drosophila melanogaster/genética , Regulación de la Expresión Génica/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducción/genéticaRESUMEN
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.