RESUMEN
The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.
Asunto(s)
Cromosomas , ADN-Topoisomerasas de Tipo II , ADN-Topoisomerasas de Tipo II/genética , Cromosomas/genética , Mitosis/genética , Interfase/genética , PolímerosRESUMEN
Glial cells are exquisitely sensitive to neuronal injury but mechanisms by which glia establish competence to respond to injury, continuously gauge neuronal health, and rapidly activate reactive responses remain poorly defined. Here, we show glial PI3K signaling in the uninjured brain regulates baseline levels of Draper, a receptor essential for Drosophila glia to sense and respond to axonal injury. After injury, Draper levels are up-regulated through a Stat92E-modulated, injury-responsive enhancer element within the draper gene. Surprisingly, canonical JAK/STAT signaling does not regulate draper expression. Rather, we find injury-induced draper activation is downstream of the Draper/Src42a/Shark/Rac1 engulfment signaling pathway. Thus, PI3K signaling and Stat92E are critical in vivo regulators of glial responsiveness to axonal injury. We provide evidence for a positive auto-regulatory mechanism whereby signaling through the injury-responsive Draper receptor leads to Stat92E-dependent, transcriptional activation of the draper gene. We propose that Drosophila glia use this auto-regulatory loop as a mechanism to adjust their reactive state following injury.
Asunto(s)
Lesión Axonal Difusa/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , Neuroglía/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Factores de Transcripción STAT/metabolismo , Animales , Axones/metabolismo , Encéfalo/metabolismo , Drosophila , Proteínas de Drosophila/genética , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Quinasas Janus/metabolismo , Proteínas de la Membrana/genética , Neuronas Receptoras Olfatorias/fisiología , Transducción de SeñalRESUMEN
Maintaining long, energetically demanding axons throughout the life of an animal is a major challenge for the nervous system. Specialized glia ensheathe axons and support their function and integrity throughout life, but glial support mechanisms remain poorly defined. Here, we identified a collection of secreted and transmembrane molecules required in glia for long-term axon survival in vivo. We showed that the majority of components of the TGFß superfamily are required in glia for sensory neuron maintenance but not glial ensheathment of axons. In the absence of glial TGFß signaling, neurons undergo age-dependent degeneration that can be rescued either by genetic blockade of Wallerian degeneration or caspase-dependent death. Blockade of glial TGFß signaling results in increased ATP in glia that can be mimicked by enhancing glial mitochondrial biogenesis or suppressing glial monocarboxylate transporter function. We propose that glial TGFß signaling supports axon survival and suppresses neurodegeneration through promoting glial metabolic support of neurons.
Asunto(s)
Axones , Neuroglía , Factor de Crecimiento Transformador beta , Animales , Axones/metabolismo , Neuroglía/metabolismo , Nervios Periféricos/citología , Células Receptoras Sensoriales , Factor de Crecimiento Transformador beta/metabolismo , Drosophila melanogaster , Biogénesis de Organelos , Transportadores de Ácidos Monocarboxílicos/metabolismoRESUMEN
Chromosome folding is modulated as cells progress through the cell cycle. During mitosis, condensins fold chromosomes into helical loop arrays. In interphase, the cohesin complex generates loops and topologically associating domains (TADs), while a separate process of compartmentalization drives segregation of active and inactive chromatin. We used synchronized cell cultures to determine how the mitotic chromosome conformation transforms into the interphase state. Using high-throughput chromosome conformation capture (Hi-C) analysis, chromatin binding assays and immunofluorescence, we show that, by telophase, condensin-mediated loops are lost and a transient folding intermediate is formed that is devoid of most loops. By cytokinesis, cohesin-mediated CTCF-CTCF loops and the positions of TADs emerge. Compartment boundaries are also established early, but long-range compartmentalization is a slow process and proceeds for hours after cells enter G1. Our results reveal the kinetics and order of events by which the interphase chromosome state is formed and identify telophase as a critical transition between condensin- and cohesin-driven chromosome folding.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Complejos Multiproteicos/genética , Telofase , Adenosina Trifosfatasas/metabolismo , Compartimento Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Transformada , Cromatina/ultraestructura , Proteínas Cromosómicas no Histona/metabolismo , Mapeo Cromosómico , Citocinesis/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Células HeLa , Humanos , Interfase , Complejos Multiproteicos/metabolismo , Fase S , CohesinasRESUMEN
BACKGROUND: Receptor tyrosine phosphatases (RPTPs) are essential for axon guidance and synaptogenesis in Drosophila. Each guidance decision made by embryonic motor axons during outgrowth to their muscle targets requires a specific subset of the five neural RPTPs. The logic underlying these requirements, however, is still unclear, partially because the ligands recognized by RPTPs at growth cone choice points have not been identified. RPTPs in general are still "orphan receptors" because, while they have been found to interact in vitro with many different proteins, their in vivo ligands are unknown. RESULTS: Here we use a new type of deficiency screen to identify the transmembrane heparan sulfate proteoglycan Syndecan (Sdc) as a ligand for the neuronal RPTP LAR. LAR interacts with the glycosaminoglycan chains of Syndecan in vitro with nanomolar affinity. Genetic interaction studies using Sdc and Lar LOF mutations demonstrate that Sdc contributes to LAR's function in motor axon guidance. We also show that overexpression of Sdc on muscles generates the same phenotype as overexpression of LAR in neurons and that genetic removal of LAR suppresses the phenotype produced by ectopic muscle Sdc. Finally, we show that there is at least one additional, nonproteoglycan, ligand for LAR encoded in the genome. CONCLUSIONS: Taken together, our results demonstrate that Sdc on muscles can interact with neuronal LAR in vivo and that binding to Sdc increases LAR's signaling efficacy. Thus, Sdc is a ligand that can act in trans to positively regulate signal transduction through LAR within neuronal growth cones.
Asunto(s)
Drosophila/metabolismo , Conos de Crecimiento/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteoglicanos/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/genética , Animales , Línea Celular , Cruzamientos Genéticos , Drosophila/genética , Drosophila/fisiología , Conos de Crecimiento/fisiología , Proteoglicanos de Heparán Sulfato/genética , Humanos , Inmunohistoquímica , Inmunoprecipitación , Glicoproteínas de Membrana/genética , Unión Proteica , Proteoglicanos/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , SindecanosRESUMEN
The NAD-synthesising enzyme Nmnat2 is a critical survival factor for axons in vitro and in vivo. We recently reported that loss of axonal transport vesicle association through mutations in its isoform-specific targeting and interaction domain (ISTID) reduces Nmnat2 ubiquitination, prolongs its half-life and boosts its axon protective capacity in primary culture neurons. Here, we report evidence for a role of ISTID sequences in tuning Nmnat2 localisation, stability and protective capacity in vivo. Deletion of central ISTID sequences abolishes vesicle association and increases protein stability of fluorescently tagged, transgenic Nmnat2 in mouse peripheral axons in vivo. Overexpression of fluorescently tagged Nmnat2 significantly delays Wallerian degeneration in these mice. Furthermore, while mammalian Nmnat2 is unable to protect transected Drosophila olfactory receptor neuron axons in vivo, mutant Nmnat2s lacking ISTID regions substantially delay Wallerian degeneration. Together, our results establish Nmnat2 localisation and turnover as a valuable target for modulating axon degeneration in vivo.
Asunto(s)
Axones/metabolismo , Axones/patología , Eliminación de Gen , Nicotinamida-Nucleótido Adenililtransferasa/genética , Fracciones Subcelulares/metabolismo , Degeneración Walleriana/patología , Degeneración Walleriana/fisiopatología , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Relación Estructura-Actividad , Fracciones Subcelulares/ultraestructura , Degeneración Walleriana/prevención & controlRESUMEN
Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Here, we show that loss of the Drosophila Toll receptor adaptor dSarm (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway.
Asunto(s)
Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/fisiología , Axones/fisiología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Neuronas/fisiología , Degeneración Walleriana , Animales , Animales Modificados Genéticamente , Apoptosis , Proteínas del Dominio Armadillo/análisis , Axones/ultraestructura , Axotomía , Supervivencia Celular , Células Cultivadas , Proteínas del Citoesqueleto/análisis , Desnervación , Drosophila/embriología , Drosophila/genética , Drosophila/fisiología , Proteínas de Drosophila/análisis , Ratones , Mutación , Nervio Ciático/lesiones , Nervio Ciático/fisiología , Transducción de Señal , Ganglio Cervical Superior/citología , Técnicas de Cultivo de TejidosRESUMEN
This paper defines a collection of Drosophila deletion mutations (deficiencies) that can be systematically screened for embryonic phenotypes, orphan receptor ligands, and genes affecting protein localization. It reports the results of deficiency screens we have conducted that have revealed new axon guidance phenotypes in the central nervous system and neuromuscular system and permitted a quantitative assessment of the number of potential genes involved in regulating guidance of specific motor axon branches. Deficiency "kits" that cover the genome with a minimum number of lines have been established to facilitate gene mapping. These kits cannot be systematically analyzed for phenotypes, however, since embryos homozygous for many deficiencies in these kits fail to develop due to the loss of key gene products encoded within the deficiency. To create new kits that can be screened for phenotype, we have examined the development of the nervous system in embryos homozygous for more than 700 distinct deficiency mutations. A kit of approximately 400 deficiency lines for which homozygotes have a recognizable nervous system and intact body walls encompasses >80% of the genome. Here we show examples of screens of this kit for orphan receptor ligands and neuronal antigen expression. It can also be used to find genes involved in expression, patterning, and subcellular localization of any protein that can be visualized by antibody staining. A subset kit of 233 deficiency lines, for which homozygotes develop relatively normally to late stage 16, covers approximately 50% of the genome. We have screened it for axon guidance phenotypes, and we present examples of new phenotypes we have identified. The subset kit can be used to screen for phenotypes affecting all embryonic organs. In the future, these deficiency kits will allow Drosophila researchers to rapidly and efficiently execute genome-wide anatomical screens that require examination of individual embryos at high magnification.
Asunto(s)
Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Genes de Insecto/genética , Ligandos , Fenotipo , Receptores de Superficie Celular/metabolismo , Eliminación de Secuencia , Animales , Antígenos/metabolismo , Axones/metabolismo , Sistema Nervioso Central/citología , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/metabolismo , Regulación de la Expresión Génica , Humanos , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Neuroglía/metabolismo , Factores de TiempoRESUMEN
Anopheles gambiae is a highly anthropophilic mosquito responsible for the majority of malaria transmission in Africa. The biting and host preference behavior of this disease vector is largely influenced by its sense of smell, which is presumably facilitated by G protein-coupled receptor signaling [Takken, W. & Knols, B. (1999) Annu. Rev. Entomol. 44, 131-157]. Because of the importance of host preference to the mosquitoes' ability to transmit disease, we have initiated studies intended to elucidate the molecular mechanisms underlying olfaction in An. gambiae. In the course of these studies, we have identified a number of genes potentially involved in signal transduction, including a family of candidate odorant receptors. One of these receptors, encoded by GPRor7 (hereafter referred to as AgOr7), is remarkably similar to an odorant receptor that is expressed broadly in olfactory tissues and has been identified in Drosophila melanogaster and other insects [Krieger, J., Klink, O., Mohl, C., Raming, K. & Breer, H. (2003) J. Comp. Physiol. A 189, 519-526; Vosshall, L. B., Amrein, H., Morozov, P. S., Rzhetsky, A. & Axel, R. (1999) Cell 96, 725-736]. We have observed AgOr7 expression in olfactory and gustatory tissues in adult An. gambiae and during several stages of the mosquitoes' development. Within the female adult peripheral chemosensory system, antiserum against the AgOR7 polypeptide labels most sensilla of the antenna and maxillary palp as well as a subset of proboscis sensilla. Furthermore, AgOR7 antiserum labeling is observed within the larval antenna and maxillary palpus. These results are consistent with a role for AgOr7 in both olfaction and gustation in An. gambiae and raise the possibility that AgOr7 orthologs may also be of general importance to both modalities of chemosensation in other insects.
Asunto(s)
Anopheles/metabolismo , Células Quimiorreceptoras/metabolismo , Olfato , Gusto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Quimiorreceptoras/química , Cromosomas Artificiales Bacterianos , Cartilla de ADN , Femenino , Inmunohistoquímica , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Receptores de Superficie Celular , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
We used bioinformatic approaches to identify a total of 276 G protein-coupled receptors (GPCRs) from the Anopheles gambiae genome. These include GPCRs that are likely to play roles in pathways affecting almost every aspect of the mosquito's life cycle. Seventy-nine candidate odorant receptors were characterized for tissue expression and, along with 76 putative gustatory receptors, for their molecular evolution relative to Drosophila melanogaster. Examples of lineage-specific gene expansions were observed as well as a single instance of unusually high sequence conservation.