RESUMO
Temperature influences animal physiology and behavior. Animals must set an appropriate body temperature to maintain homeostasis and maximize survival. Mammals set their body temperatures using metabolic and behavioral strategies. The daily fluctuation in body temperature is called the body temperature rhythm (BTR). For example, human body temperature increases during wakefulness and decreases during sleep. BTR is controlled by the circadian clock, is closely linked with metabolism and sleep, and entrains peripheral clocks located in the liver and lungs. However, the underlying mechanisms of BTR are largely unclear. In contrast to mammals, small ectotherms, such as Drosophila, control their body temperatures by choosing appropriate environmental temperatures. The preferred temperature of Drosophila increases during the day and decreases at night; this pattern is referred to as the temperature preference rhythm (TPR). As flies are small ectotherms, their body temperature is close to that of the surrounding environment. Thus, Drosophila TPR produces BTR, which exhibits a pattern similar to that of human BTR. In this review, we summarize the regulatory mechanisms of TPR, including recent studies that describe neuronal circuits relaying ambient temperature information to dorsal neurons (DNs). The neuropeptide diuretic hormone 31 (DH31) and its receptor (DH31R) regulate TPR, and a mammalian homolog of DH31R, the calcitonin receptor (CALCR), also plays an important role in mouse BTR regulation. In addition, both fly TPR and mammalian BTR are separately regulated from another clock output, locomotor activity rhythms. These findings suggest that the fundamental mechanisms of BTR regulation may be conserved between mammals and flies. Furthermore, we discuss the relationships between TPR and other physiological functions, such as sleep. The dissection of the regulatory mechanisms of Drosophila TPR could facilitate an understanding of mammalian BTR and the interaction between BTR and sleep regulation.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Humanos , Animais , Camundongos , Drosophila melanogaster/fisiologia , Temperatura , Proteínas de Drosophila/metabolismo , Ritmo Circadiano/fisiologia , Drosophila/fisiologia , Mamíferos/metabolismoRESUMO
Animals display a body temperature rhythm (BTR). Little is known about the mechanisms by which a rhythmic pattern of BTR is regulated and how body temperature is set at different times of the day. As small ectotherms, Drosophila exhibit a daily temperature preference rhythm (TPR), which generates BTR. Here, we demonstrate dorsal clock networks that play essential roles in TPR. Dorsal neurons 2 (DN2s) are the main clock for TPR. We find that DN2s and posterior DN1s (DN1ps) contact and the extent of contacts increases during the day and that the silencing of DN2s or DN1ps leads to a lower temperature preference. The data suggest that temporal control of the microcircuit from DN2s to DN1ps contributes to TPR regulation. We also identify anterior DN1s (DN1as) as another important clock for TPR. Thus, we show that the DN networks predominantly control TPR and determine both a rhythmic pattern and preferred temperatures.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Ritmo Circadiano/fisiologia , Drosophila/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Neurônios/fisiologia , TemperaturaRESUMO
Human body temperature increases during wakefulness and decreases during sleep. The body temperature rhythm (BTR) is a robust output of the circadian clock and is fundamental for maintaining homeostasis, such as generating metabolic energy and sleep, as well as entraining peripheral clocks in mammals. However, the mechanisms that regulate BTR are largely unknown. Drosophila are ectotherms, and their body temperatures are close to ambient temperature; therefore, flies select a preferred environmental temperature to set their body temperature. We identified a novel circadian output, the temperature preference rhythm (TPR), in which the preferred temperature in flies increases during the day and decreases at night. TPR, thereby, produces a daily BTR. We found that fly TPR shares many features with mammalian BTR. We demonstrated that diuretic hormone 31 receptor (DH31R) mediates Drosophila TPR and that the closest mouse homolog of DH31R, calcitonin receptor (Calcr), is essential for mice BTR. Importantly, both TPR and BTR are regulated in a distinct manner from locomotor activity rhythms, and neither DH31R nor Calcr regulates locomotor activity rhythms. Our findings suggest that DH31R/Calcr is an ancient and specific mediator of BTR. Thus, understanding fly TPR will provide fundamental insights into the molecular and neural mechanisms that control BTR in mammals.
Assuntos
Ritmo Circadiano , Drosophila/fisiologia , Animais , Temperatura Corporal , Regulação da Temperatura Corporal , Proteínas de Drosophila/metabolismo , Humanos , Locomoção , Neurônios/metabolismo , Receptores da Calcitonina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , TemperaturaRESUMO
Neuropeptides play pivotal roles in modulating circadian rhythms. Pigment-dispersing factor (PDF) is critical to the circadian rhythms in Drosophila locomotor activity. Here, we demonstrate that diuretic hormone 31 (DH31) complements PDF function in regulating free-running rhythmicity using male flies. We determined that Dh31 loss-of-function mutants (Dh31#51) showed normal rhythmicity, whereas Dh31#51;Pdf01 double mutants exhibited a severe arrhythmic phenotype compared to Pdf-null mutants (Pdf01). The expression of tethered-PDF or tethered-DH31 in clock cells, posterior dorsal neurons 1 (DN1ps), overcomes the severe arrhythmicity of Dh31#51;Pdf01 double mutants, suggesting that DH31 and PDF may act on DN1ps to regulate free-running rhythmicity in a hierarchical manner. Unexpectedly, the molecular oscillations in Dh31#51;Pdf01 mutants were similar to those in Pdf01 mutants in DN1ps, indicating that DH31 does not contribute to molecular oscillations. Furthermore, a reduction in Dh31 receptor (Dh31r) expression resulted in normal locomotor activity and did not enhance the arrhythmic phenotype caused by the Pdf receptor (Pdfr) mutation, suggesting that PDFR, but not DH31R, in DN1ps mainly regulates free-running rhythmicity. Taken together, we identify a novel role of DH31, in which DH31 and PDF hierarchically regulate free-running rhythmicity through DN1ps.
Assuntos
Ritmo Circadiano/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Hormônios de Inseto/metabolismo , Locomoção/fisiologia , Neuropeptídeos/metabolismo , Animais , Relógios Biológicos/fisiologia , Drosophila/genética , Proteínas de Drosophila/genética , Deleção de Genes , Hormônios de Inseto/genética , Masculino , Atividade Motora/fisiologia , Neuropeptídeos/genéticaRESUMO
Starvation is life-threatening and therefore strongly modulates many aspects of animal behavior and physiology [1]. In mammals, hunger causes a reduction in body temperature and metabolism [2], resulting in conservation of energy for survival. However, the molecular basis of the modulation of thermoregulation by starvation remains largely unclear. Whereas mammals control their body temperature internally, small ectotherms, such as Drosophila, set their body temperature by selecting an ideal environmental temperature through temperature preference behaviors [3, 4]. Here, we demonstrate in Drosophila that starvation results in a lower preferred temperature, which parallels the reduction in body temperature in mammals. The insulin/insulin-like growth factor (IGF) signaling (IIS) pathway is involved in starvation-induced behaviors and physiology and is well conserved in vertebrates and invertebrates [5-7]. We show that insulin-like peptide 6 (Ilp6) in the fat body (fly liver and adipose tissues) is responsible for the starvation-induced reduction in preferred temperature (Tp). Temperature preference behavior is controlled by the anterior cells (ACs), which respond to warm temperatures via transient receptor potential A1 (TrpA1) [4]. We demonstrate that starvation decreases the responding temperature of ACs via insulin signaling, resulting in a lower Tp than in nutrient-rich conditions. Thus, we show that hunger information is conveyed from fat tissues via Ilp6 and influences the sensitivity of warm-sensing neurons in the brain, resulting in a lower temperature set point. Because starvation commonly results in a lower body temperature in both flies and mammals, we propose that insulin signaling is an ancient mediator of starvation-induced thermoregulation.
Assuntos
Regulação da Temperatura Corporal/fisiologia , Drosophila melanogaster/fisiologia , Neurônios/fisiologia , Transdução de Sinais , Animais , Comportamento Alimentar , Insulina , MotivaçãoRESUMO
Daily body temperature rhythm (BTR) is essential for maintaining homeostasis. BTR is regulated separately from locomotor activity rhythms, but its molecular basis is largely unknown. While mammals internally regulate BTR, ectotherms, including Drosophila, exhibit temperature preference rhythm (TPR) behavior to regulate BTR. Here, we demonstrate that the diuretic hormone 31 receptor (DH31R) mediates TPR during the active phase in Drosophila DH31R is expressed in clock cells, and its ligand, DH31, acts on clock cells to regulate TPR during the active phase. Surprisingly, the mouse homolog of DH31R, calcitonin receptor (Calcr), is expressed in the suprachiasmatic nucleus (SCN) and mediates body temperature fluctuations during the active phase in mice. Importantly, DH31R and Calcr are not required for coordinating locomotor activity rhythms. Our results represent the first molecular evidence that BTR is regulated distinctly from locomotor activity rhythms and show that DH31R/Calcr is an ancient specific mediator of BTR during the active phase in organisms ranging from ectotherms to endotherms.
Assuntos
Regulação da Temperatura Corporal , Proteínas de Drosophila/fisiologia , Receptores da Calcitonina/fisiologia , Animais , Encéfalo/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Hormônios de Inseto/fisiologia , Locomoção , Camundongos , Mutação , Neuropeptídeos/fisiologia , Receptores da Calcitonina/metabolismo , Núcleo Supraquiasmático/metabolismoRESUMO
Animals have sophisticated homeostatic controls. While mammalian body temperature fluctuates throughout the day, small ectotherms, such as Drosophila achieve a body temperature rhythm (BTR) through their preference of environmental temperature. Here, we demonstrate that pigment dispersing factor (PDF) neurons play an important role in setting preferred temperature before dawn. We show that small lateral ventral neurons (sLNvs), a subset of PDF neurons, activate the dorsal neurons 2 (DN2s), the main circadian clock cells that regulate temperature preference rhythm (TPR). The number of temporal contacts between sLNvs and DN2s peak before dawn. Our data suggest that the thermosensory anterior cells (ACs) likely contact sLNvs via serotonin signaling. Together, the ACs-sLNs-DN2s neural circuit regulates the proper setting of temperature preference before dawn. Given that sLNvs are important for sleep and that BTR and sleep have a close temporal relationship, our data highlight a possible neuronal interaction between body temperature and sleep regulation.
Assuntos
Temperatura Corporal , Drosophila/fisiologia , Rede Nervosa/fisiologia , Neurônios/fisiologia , Animais , Relógios Circadianos , Drosophila/efeitos da radiação , Proteínas de Drosophila/metabolismo , Homeostase , Neurônios/química , Neuropeptídeos/metabolismo , Serotonina/metabolismoRESUMO
Body temperature exhibits rhythmic fluctuations over a 24 h period (Refinetti and Menaker, 1992) and decreases during the night, which is associated with sleep initiation (Gilbert et al., 2004; Kräuchi, 2007a,b). However, the underlying mechanism of this temperature decrease is largely unknown. We have previously shown that Drosophila exhibit a daily temperature preference rhythm (TPR), in which their preferred temperatures increase during the daytime and then decrease at the transition from day to night (night-onset) (Kaneko et al., 2012). Because Drosophila are small ectotherms, their body temperature is very close to that of the ambient temperature (Stevenson, 1985), suggesting that their TPR generates their body temperature rhythm. Here, we demonstrate that the neuropeptide diuretic hormone 31 (DH31) and pigment-dispersing factor receptor (PDFR) contribute to regulate the preferred temperature decrease at night-onset. We show that PDFR and tethered-DH31 expression in dorsal neurons 2 (DN2s) restore the preferred temperature decrease at night-onset, suggesting that DH31 acts on PDFR in DN2s. Notably, we previously showed that the molecular clock in DN2s is important for TPR. Although PDF (another ligand of PDFR) is a critical factor for locomotor activity rhythms, Pdf mutants exhibit normal preferred temperature decreases at night-onset. This suggests that DH31-PDFR signaling specifically regulates a preferred temperature decrease at night-onset. Thus, we propose that night-onset TPR and locomotor activity rhythms are differentially controlled not only by clock neurons but also by neuropeptide signaling in the brain. SIGNIFICANCE STATEMENT: Body temperature rhythm (BTR) is fundamental for the maintenance of functions essential for homeostasis, such as generating metabolic energy and sleep. One major unsolved question is how body temperature decreases dramatically during the night. Previously, we demonstrated that a BTR-like mechanism, referred to as temperature preference rhythm (TPR), exists in Drosophila Here, we demonstrate that the diuretic hormone 31 (DH31) neuropeptide and pigment-dispersing factor receptor (PDFR) regulate preferred temperature decreases at night-onset via dorsal neurons 2. This is the first in vivo evidence that DH31 could function as a ligand of PDFR. Although both DH31 and PDF are ligands of PDFR, we show that DH31 regulates night-onset TPR, but PDF does not, suggesting that night-onset TPR and locomotor activity rhythms are controlled by different neuropeptides via different clock cells.
Assuntos
Regulação da Temperatura Corporal/fisiologia , Ritmo Circadiano/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Hormônios de Inseto/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriais/fisiologia , Animais , Relógios Biológicos/fisiologia , Regulação da Expressão GênicaRESUMO
Ambient light affects multiple physiological functions and behaviors, such as circadian rhythms, sleep-wake activities, and development, from flies to mammals. Mammals exhibit a higher body temperature when exposed to acute light compared to when they are exposed to the dark, but the underlying mechanisms are largely unknown. The body temperature of small ectotherms, such as Drosophila, relies on the temperature of their surrounding environment, and these animals exhibit a robust temperature preference behavior. Here, we demonstrate that Drosophila prefer a â¼1° higher temperature when exposed to acute light rather than the dark. This acute light response, light-dependent temperature preference (LDTP), was observed regardless of the time of day, suggesting that LDTP is regulated separately from the circadian clock. However, screening of eye and circadian clock mutants suggests that the circadian clock neurons posterior dorsal neurons 1 (DN1(p)s) and Pigment-Dispersing Factor Receptor (PDFR) play a role in LDTP. To further investigate the role of DN1(p)s in LDTP, PDFR in DN1(p)s was knocked down, resulting in an abnormal LDTP. The phenotype of the pdfr mutant was rescued sufficiently by expressing PDFR in DN1(p)s, indicating that PDFR in DN1(p)s is responsible for LDTP. These results suggest that light positively influences temperature preference via the circadian clock neurons, DN1(p)s, which may result from the integration of light and temperature information. Given that both Drosophila and mammals respond to acute light by increasing their body temperature, the effect of acute light on temperature regulation may be conserved evolutionarily between flies and humans.
Assuntos
Relógios Circadianos/fisiologia , Proteínas de Drosophila/metabolismo , Luz , Neurônios/citologia , Receptores Acoplados a Proteínas G/metabolismo , Núcleo Supraquiasmático/citologia , Temperatura , Animais , Ritmo Circadiano/fisiologia , Drosophila , Neurônios/metabolismoRESUMO
The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms(1) (,) (2). We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night (3). Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons(3). Drosophila locomotor activity is a well known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms(4). Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR(3). Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies(5-8). We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.
Assuntos
Comportamento Animal/fisiologia , Regulação da Temperatura Corporal/fisiologia , Ritmo Circadiano/fisiologia , Drosophila/fisiologia , Animais , Locomoção/fisiologia , TemperaturaRESUMO
Temperature sensation has a strong impact on animal behavior and is necessary for animals to avoid exposure to harmful temperatures. It is now well known that thermoTRP (transient receptor potential) channels in thermosensory neurons detect a variable range of temperature stimuli. However, little is known about how a range of temperature information is relayed and integrated in the neural circuits. Here, we show novel temperature integration between two warm inputs via Drosophila TRPA channels, TRPA1 and Pyrexia (Pyx). The internal AC (anterior cell) thermosensory neurons, which express TRPA1, detect warm temperatures and mediate temperature preference behavior. We found that the AC neurons were activated twice when subjected to increasing temperatures. The first response was at â¼25°C via TRPA1 channel, which is expressed in the AC neurons. The second response was at â¼27°C via the second antennal segments, indicating that the second antennal segments are involved in the detection of warm temperatures. Further analysis reveals that pyx-Gal4-expressing neurons have synapses on the AC neurons and that mutation of pyx eliminates the second response of the AC neurons. These data suggest that AC neurons integrate both their own TRPA1-dependent temperature responses and a Pyx-dependent temperature response from the second antennal segments. Our data reveal the first identification of temperature integration, which combines warm temperature information from peripheral to central neurons and provides the possibility that temperature integration is involved in the plasticity of behavioral outputs.
Assuntos
Encéfalo/fisiologia , Proteínas de Drosophila/metabolismo , Neurônios/fisiologia , Canais de Cátion TRPC/metabolismo , Sensação Térmica/fisiologia , Animais , Antenas de Artrópodes/fisiologia , Drosophila , Proteínas de Drosophila/genética , Canais Iônicos , Vias Neurais/fisiologia , Canal de Cátion TRPA1 , Canais de Cátion TRPC/genética , TemperaturaRESUMO
A daily body temperature rhythm (BTR) is critical for the maintenance of homeostasis in mammals. Whereas mammals use internal energy to regulate body temperature, ectotherms typically regulate body temperature behaviorally [1]. Some ectotherms maintain homeostasis via a daily temperature preference rhythm (TPR) [2], but the underlying mechanisms are largely unknown. Here, we show that Drosophila exhibit a daily circadian clock-dependent TPR that resembles mammalian BTR. Pacemaker neurons critical for locomotor activity are not necessary for TPR; instead, the dorsal neuron 2 s (DN2s), whose function was previously unknown, is sufficient. This indicates that TPR, like BTR, is controlled independently from locomotor activity. Therefore, the mechanisms controlling temperature fluctuations in fly TPR and mammalian BTR may share parallel features. Taken together, our results reveal the existence of a novel DN2-based circadian neural circuit that specifically regulates TPR; thus, understanding the mechanisms of TPR will shed new light on the function and neural control of circadian rhythms.
Assuntos
Ritmo Circadiano/fisiologia , Drosophila/fisiologia , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Neurônios/fisiologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , TemperaturaRESUMO
Mer3 is an evolutionarily conserved DNA helicase that has crucial roles in meiotic recombination and crossover formation. We have identified the MER3 homolog in Coprinus cinereus (Ccmer3) and show that it is expressed in zygotene and pachytene meiocytes. Immunostaining analysis indicated that CcMer3 was localized on chromosomes at zygotene and pachytene and CcMer3 foci were more frequent on paired than unpaired chromosomes. We generated a C. cinereus mer3 mutant (#1) and found that it showed abnormal meiosis progression and underwent apoptosis after prophase I. Basidiospore production in #1 was reduced to 0.8% of the wild-type level; the spores showed slower germination at 25 degrees C but were similar to the wild type at 37 degrees C. Electron microscopic analysis of chromosome spreads revealed that axial elements were formed in the mutant but that synapsis was defective, resulting in a reduction in spore production. Our results demonstrate that CcMer3 is required for synaptonemal complex formation after axial elements align and is thus essential for homologous synapsis.
Assuntos
Coprinus/genética , Coprinus/metabolismo , DNA Helicases/metabolismo , Genes Fúngicos/fisiologia , Meiose/fisiologia , Complexo Sinaptonêmico/metabolismo , DNA Helicases/genética , Genes Fúngicos/genética , Meiose/genética , Microscopia , Proteínas Recombinantes/metabolismoRESUMO
Animals from flies to humans are able to distinguish subtle gradations in temperature and show strong temperature preferences. Animals move to environments of optimal temperature and some manipulate the temperature of their surroundings, as humans do using clothing and shelter. Despite the ubiquitous influence of environmental temperature on animal behaviour, the neural circuits and strategies through which animals select a preferred temperature remain largely unknown. Here we identify a small set of warmth-activated anterior cell (AC) neurons located in the Drosophila brain, the function of which is critical for preferred temperature selection. AC neuron activation occurs just above the fly's preferred temperature and depends on dTrpA1, an ion channel that functions as a molecular sensor of warmth. Flies that selectively express dTrpA1 in the AC neurons select normal temperatures, whereas flies in which dTrpA1 function is reduced or eliminated choose warmer temperatures. This internal warmth-sensing pathway promotes avoidance of slightly elevated temperatures and acts together with a distinct pathway for cold avoidance to set the fly's preferred temperature. Thus, flies select a preferred temperature by using a thermal sensing pathway tuned to trigger avoidance of temperatures that deviate even slightly from the preferred temperature. This provides a potentially general strategy for robustly selecting a narrow temperature range optimal for survival.
Assuntos
Comportamento de Escolha/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Canais de Cátion TRPC/metabolismo , Temperatura , Animais , Aprendizagem da Esquiva , Temperatura Corporal , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Canais Iônicos , Larva , Dados de Sequência Molecular , Neurônios/metabolismo , Oócitos/metabolismo , Canal de Cátion TRPA1 , Canais de Cátion TRPC/genética , Xenopus laevisRESUMO
In eukaryotes, meiosis leads to genetically variable gametes through recombination between homologous chromosomes of maternal and paternal origin. Chromatin organization following meiotic recombination is critical to ensure the correct segregation of homologous chromosomes into gametes. However, the mechanism of chromatin organization after meiotic recombination is unknown. In this study we report that the meiosis-specific recombinase Lim15/Dmc1 interacts with the homologue of the largest subunit of chromatin assembly factor 1 (CAF-1) in the basidiomycete Coprinopsis cinerea (Coprinus cinereus). Using C. cinerea LIM15/DMC1 (CcLIM15) as the bait in a yeast two-hybrid screen, we have isolated the C. cinerea homologue of Cac1, the largest subunit of CAF-1 in Saccharomyces cerevisiae, and named it C. cinerea Cac1-like (CcCac1L). Two-hybrid assays confirmed that CcCac1L binds CcLim15 in vivo. beta-Galactosidase assays revealed that the N-terminus of CcCac1L preferentially interacts with CcLim15. Co-immunoprecipitation experiments showed that these proteins also interact in the crude extract of meiotic cells. Furthermore, we demonstrate that, during meiosis, CcCac1L interacts with proliferating cell nuclear antigen (PCNA), a component of the DNA synthesis machinery recently reported as an interacting partner of Lim15/Dmc1. Taken together, these results suggest a novel role of the CAF-1-PCNA complex in meiotic events. We propose that the CAF-1-PCNA complex modulates chromatin assembly following meiotic recombination.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose , Recombinação Genética , Fator 1 de Modelagem da Cromatina , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Coprinus/enzimologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Modelos Biológicos , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Subunidades Proteicas/química , Ressonância de Plasmônio de Superfície , Técnicas do Sistema de Duplo-HíbridoRESUMO
PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg(2+) ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Coprinus/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose , Antígeno Nuclear de Célula em Proliferação/metabolismo , Recombinases Rec A/metabolismo , Recombinação Genética/genética , Animais , Proteínas de Ciclo Celular/genética , Coprinus/citologia , Coprinus/genética , Proteínas de Ligação a DNA/genética , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica , Recombinases Rec A/genéticaRESUMO
Sumoylation is a post-translational modification system that covalently attaches the small ubiquitin-related modifier (SUMO) to target proteins. Ubc9 is required as the E2-type enzyme for SUMO-1 conjugation to targets. Here, we show that Ubc9 interacts with the meiosis-specific RecA homolog, Lim15/Dmc1 in the basidiomycete Coprinus cinereus (CcLim15), and mediates sumoylation of CcLim15 during meiosis. In vitro protein-protein interaction assays revealed that CcUbc9 interacts with CcLim15 and binds to the C-terminus (amino acids 105-347) of CcLim15, which includes the ATPase domain. Immunocytochemistry demonstrates that CcUbc9 and CcLim15 colocalize in the nuclei from the leptotene stage to the early pachytene stage during meiotic prophase I. Coimmunoprecipitation experiments indicate that CcUbc9 interacts with CcLim15 in vivo during meiotic prophase I. Furthermore, we show that CcLim15 is a target protein of sumoylation both in vivo and in vitro, and identify the C-terminus (amino acids 105-347) of CcLim15 as the site of sumoylation in vitro. These results suggest that sumoylation is a candidate modulator of meiotic recombination via interaction between Ubc9 and Lim15/Dmc1.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Meiose/fisiologia , Recombinases Rec A/química , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Enzimas de Conjugação de Ubiquitina/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Agaricales/citologia , Agaricales/enzimologia , Proteínas de Ciclo Celular/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/química , Dados de Sequência Molecular , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The basidiomycete Coprinus cinereus has many advantages as a model organism for studying sexual development and meiosis, but it has been difficult to investigate using reverse-genetics methods, such as gene disruption by homologous recombination. Here, gene repression by dsRNA-mediated gene silencing was tried as an alternative method for reverse-genetics studies. It was shown that transformation of the LIM15/DMC1 dsRNA expression construct (LIM15dsRNA) resulted in genomic insertion of LIM15dsRNA and paucity of the LIM15/DMC1 transcript. First, LIM15dsRNA was transformed into the homothallic strain AmutBmut to generate a homozygote in which both nuclei had a copy of LIM15dsRNA. The LIM15/DMC1-repressed strain showed abnormal homologous chromosome synapsis during meiosis. Basidiospore production was reduced to 16 % by the induction of dsRNA. However, approximately 60 % of basidiospores were viable. Next, a heterozygote was generated in which one nucleus had a copy of LIM15dsRNA. The phenotype was similar to that of the homozygote. These results are not only the first demonstration of dsRNA-mediated gene silencing in a member of the homobasidiomycete fungi, to which 90 % of mushroom species belong, but also the first successful use of a reverse-genetics approach in C. cinereus research.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Coprinus/fisiologia , Proteínas de Ligação a DNA/genética , Inativação Gênica , Meiose , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Coprinus/genética , Coprinus/crescimento & desenvolvimento , Coprinus/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA de Cadeia Dupla/genética , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Lim15/Dmc1 is a meiosis specific RecA-like protein. Here we propose its participation in meiotic chromosome pairing-related events along with DNA topoisomerase II. Analysis of protein-protein interactions using in vitro binding assays provided evidence that Coprinus cinereus DNA topoisomerase II (CcTopII) specifically interacts with C.cinereus Lim15/Dmc1 (CcLim15). Co-immunoprecipitation experiments also indicated that the CcLim15 protein interacts with CcTopII in vivo. Furthermore, a significant proportion of CcLim15 and CcTopII could be shown to co-localize on chromosomes from the leptotene to the zygotene stage. Interestingly, CcLim15 can potently activate the relaxation/catenation activity of CcTopII in vitro, and CcTopII suppresses CcLim15-dependent strand transfer activity. On the other hand, while enhancement of CcLim15's DNA-dependent ATPase activity by CcTopII was found in vitro, the same enzyme activity of CcTopII was inhibited by adding CcLim15. The interaction of CcLim15 and CcTopII may facilitate pairing of homologous chromosomes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose , Sequência de Bases , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/química , Núcleo Celular/química , Núcleo Celular/enzimologia , Coprinus/enzimologia , DNA Topoisomerases Tipo II/análise , DNA Topoisomerases Tipo II/química , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/química , Imunoprecipitação , Dados de Sequência Molecular , Deleção de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
A long-standing model postulates that X-chromosome dosage compensation in Drosophila occurs by twofold up-regulation of the single male X, but previous data cannot exclude an alternative model, in which male autosomes are down-regulated to balance gene expression. To distinguish between the two models, we used RNA interference to deplete Male-Specific Lethal (MSL) complexes from male-like tissue culture cells. We found that expression of many genes from the X chromosome decreased, while expression from the autosomes was largely unchanged. We conclude that the primary role of the MSL complex is to up-regulate the male X chromosome.