Active Toll Research Articles

Microtubule disruption upon CNS damage triggers mitotic entry via TNF signaling activation.

Repair after traumatic injury often starts with mitotic activation around the lesion edges. Early midline cells in the Drosophila embryonic CNS can enter into division following the traumatic disruption of microtubules. We demonstrate that microtubule disruption activates non-canonical TNF signaling by phosphorylation of TGF-β activated kinase 1 (Tak1) and its target IkappaB kinase (Ik2), culminating in Dorsal/NfkappaB nuclear translocation and Jra/Jun expression. Tak1 and Ik2 are necessary for the damaged-induced divisions. Microtubule disruption caused by Tau accumulation is also reported in Alzheimer's disease (AD). Human Tau expression in Drosophila midline cells is sufficient to induce Tak1 phosphorylation, Dorsal and Jra/Jun expression, and entry into mitosis. Interestingly, activation of Tak1 and Tank binding kinase 1 (Tbk1), the human Ik2 ortholog, and NfkappaB upregulation are observed in AD brains.

Innate immune signaling in Drosophila shifts anabolic lipid metabolism from triglyceride storage to phospholipid synthesis to support immune function

During infection, cellular resources are allocated toward the metabolically-demanding processes of synthesizing and secreting effector proteins that neutralize and kill invading pathogens. In Drosophila, these effectors are antimicrobial peptides (AMPs) that are produced in the fat body, an organ that also serves as a major lipid storage depot. Here we asked how activation of Toll signaling in the larval fat body perturbs lipid homeostasis to understand how cells meet the metabolic demands of the immune response. We find that genetic or physiological activation of fat body Toll signaling leads to a tissue-autonomous reduction in triglyceride storage that is paralleled by decreased transcript levels of the DGAT homolog midway, which carries out the final step of triglyceride synthesis. In contrast, Kennedy pathway enzymes that synthesize membrane phospholipids are induced. Mass spectrometry analysis revealed elevated levels of major phosphatidylcholine and phosphatidylethanolamine species in fat bodies with active Toll signaling. The ER stress mediator Xbp1 contributed to the Toll-dependent induction of Kennedy pathway enzymes, which was blunted by deleting AMP genes, thereby reducing secretory demand elicited by Toll activation. Consistent with ER stress induction, ER volume is expanded in fat body cells with active Toll signaling, as determined by transmission electron microscopy. A major functional consequence of reduced Kennedy pathway induction is an impaired immune response to bacterial infection. Our results establish that Toll signaling induces a shift in anabolic lipid metabolism to favor phospholipid synthesis and ER expansion that may serve the immediate demand for AMP synthesis and secretion but with the long-term consequence of insufficient nutrient storage.

Robustness of the Dorsal morphogen gradient with respect to morphogen dosage.

In multicellular organisms, the timing and placement of gene expression in a developing tissue assigns the fate of each cell in the embryo in order for a uniform field of cells to differentiate into a reproducible pattern of organs and tissues. This positional information is often achieved through the action of spatial gradients of morphogens. Spatial patterns of gene expression are paradoxically robust to variations in morphogen dosage, given that, by definition, gene expression must be sensitive to morphogen concentration. In this work we investigate the robustness of the Dorsal/NF-κB signaling module with respect to perturbations to the dosage of maternally-expressed dorsal mRNA. The Dorsal morphogen gradient patterns the dorsal-ventral axis of the early Drosophila embryo, and we found that an empirical description of the Dorsal gradient is highly sensitive to maternal dorsal dosage. In contrast, we found experimentally that gene expression patterns are highly robust. Although the components of this signaling module have been characterized in detail, how their function is integrated to produce robust gene expression patterns to variations in the dorsal maternal dosage is still unclear. Therefore, we analyzed a mechanistic model of the Dorsal signaling module and found that Cactus, a cytoplasmic inhibitor for Dorsal, must be present in the nucleus for the system to be robust. Furthermore, active Toll, the receptor that dissociates Cactus from Dorsal, must be saturated. Finally, the vast majority of robust descriptions of the system require facilitated diffusion of Dorsal by Cactus. Each of these three recently-discovered mechanisms of the Dorsal module are critical for robustness. These mechanisms synergistically contribute to changing the amplitude and shape of the active Dorsal gradient, which is required for robust gene expression. Our work highlights the need for quantitative understanding of biophysical mechanisms of morphogen gradients in order to understand emergent phenotypes, such as robustness.

The Toll Signaling Pathway Targets the Insulin-like Peptide Dilp6 to Inhibit Growth in Drosophila

Chronic enteropathogen infection in early childhood reduces circulating insulin-like growth factor 1 (IGF1) levels and restricts growth. Pathogen-derived molecules activate host Toll-like receptors to initiate the immune response, but whether this pathway contributes to growth inhibition is unclear. In Drosophila, activation of Toll receptors in larval fat body suppresses whole-animal growth. Here, using a transcriptomic approach, we identify Drosophila insulin-like peptide 6 (Dilp6), a fat-body-derived IGF1 ortholog, as a selective target of Toll signaling induced by infection or genetic activation of the pathway. Using a tagged allele that we generated to measure endogenous Dilp6, we find a marked reduction in circulating hormone levels. Restoring Dilp6 expression in fat body rescues growth in animals with active Toll signaling. Our results establish that Toll signaling reduces growth by inducing hormone insufficiency, implying a mechanistic link between innate immune signaling and endocrine regulation of growth.

HUBUNGAN ANTARA PERUBAHAN TUTUPAN LAHAN TERHADAP TOTAL PENDUDUK YANG DIPENGARUHI OLEH FENOMENA URBANISASI DI BOGOR, JAWA BARAT

Urbanization is the main issue that altered the environmental condition nowadays. Urbanization means that there are changes in suburban areas to fulfill the function needed in the urban area. Nowadays, urbanization occurred due to the high demand of housing area with lower budget and comfortable environment of living condition. This condition was presented in the Bogor city case. Bogor is one of the oldest cities in Indonesia which is located in the southern part of Jakarta. Due to the increase of population in the capital city and increased of the higher price of housing, people started to looked for place to stay outside the city. Bogor located in the strategic location just close enough for people to commute to Jakarta. The transit city transform into commuting city. People works in Jakarta wanted to live in a more comfortable place of living and cheaper in price than the capital city started to see Bogor as an opportunity. Based on the data obtained by the Central Bureau of Statistics Jakarta in 2015, there are 187.777 total people commuted daily from Bogor to Jakarta. This condition also triggered by the better quality of access in and out Bogor. Mass rapid transportation and highway toll gate create more land cover changes potential to fulfill the high demand of housing. Thus, this research aimed to see the effect of highway toll gate in the land use cover change in Bogor city. Landsat Images was used using the time-series method started since 1978 (the official year of Jagorawi Toll gate operation) with the 10 years interval time, 1989, 1999, 2009 and 2016. In determining the land use cover change, NDVI method was used which has been widely applied in detecting the greeneries. The result then evaluated using the supervised classification and validated using the Kappa Accuracy test. The result of this research shows that land use cover change corresponded with the total population in the citizenship census 2009. Total built area in 2009 is 36.20% which increased by 8.46% since 1999, this result suitable with the total population 949.000 in 2009 Citizenship census which increased by 364.000 since 1999. The highest built area detected is 42.15% in 2016 with total of 1.030.720 population and three active Highway toll gate operated.

Innate Immune Signaling in Drosophila Blocks Insulin Signaling by Uncoupling PI(3,4,5)P3 Production and Akt Activation

SUMMARYIn obese adipose tissue, Toll-like receptor signaling in macrophages leads to insulin resistance in adipocytes. Similarly, Toll signaling in the Drosophila larval fat body blocks insulin-dependent growth and nutrient storage. We find that Toll acts cell autonomously to block growth but not PI(3,4,5)P3 production in fat body cells expressing constitutively active PI3K. Fat body Toll signaling blocks whole-animal growth in rictor mutants lacking TORC2 activity, but not in larvae lacking Pdk1. Phosphorylation of Akt on the Pdk1 site, Thr342, is significantly reduced by Toll signaling, and expression of mutant AktT342D rescues cell and animal growth, nutrient storage, and viability in animals with active Toll signaling. Altogether, these data show that innate immune signaling blocks insulin signaling at a more distal level than previously appreciated, and they suggest that manipulations affecting the Pdk1 arm of the pathway may have profound effects on insulin sensitivity in inflamed tissues.

HUBUNGAN ANTARA PERUBAHAN TUTUPAN LAHAN TERHADAP TOTAL PENDUDUK YANG DIPENGARUHI OLEH FENOMENA URBANISASI DI BOGOR, JAWA BARAT

Urbanization is the main issue that altered the environmental condition nowadays. Urbanization means that there are changes in suburban areas to fulfill the function needed in the urban area. Nowadays, urbanization occurred due to the high demand of housing area with lower budget and comfortable environment of living condition. This condition was presented in the Bogor city case. Bogor is one of the oldest cities in Indonesia which is located in the southern part of Jakarta. Due to the increase of population in the capital city and increased of the higher price of housing , people started to looked for place to stay outside the city. Bogor located in the strategic location just close enough for people to commute to Jakarta. The transit city transform into commuting city. People works in Jakarta wanted to live in a more comfortable place of living and cheaper in price than the capital city started to see Bogor as an opportunity. Based on the data obtained by the Central Bureau of Statistics Jakarta in 2015, there are 187.777 total people commuted daily from Bogor to Jakarta. This condition also triggered by the better quality of access in and out Bogor. Mass rapid transportation and highway toll gate create more land cover changes potential to fulfill the high demand of housing. Thus, this research aimed to see the effect of highway toll gate in the land use cover change in Bogor city. Landsat Images was used using the time-series method started since 1978 (the official year of Jagorawi Toll gate operation) with the 10 years interval time, 1989, 1999, 2009 and 2016. In determining the land use cover change, NDVI method was used which has been widely applied in detecting the greeneries. The result then evaluated using the supervised classification and validated using the Kappa Accuracy test. The result of this research shows that land use cover change corresponded with the total population in the citizenship census 2009. Total built area in 2009 is 36.20% which increased by 8.46% since 1999, this result suitable with the total population 949.000 in 2009 Citizenship census which increased by 364.000 since 1999. The highest built area detected is 42.15% in 2016 with total of 1.030.720 population and three active Highway toll gate operated.

A facilitated diffusion mechanism establishes the Drosophila Dorsal gradient.

The transcription factor NF-κB plays an important role in the immune system, apoptosis and inflammation. Dorsal, a Drosophila homolog of NF-κB, patterns the dorsal-ventral axis in the blastoderm embryo. During this stage, Dorsal is sequestered outside the nucleus by the IκB homolog Cactus. Toll signaling on the ventral side breaks the Dorsal/Cactus complex, allowing Dorsal to enter the nucleus to regulate target genes. Fluorescent data show that Dorsal accumulates on the ventral side of the syncytial blastoderm. Here, we use modeling and experimental studies to show that this accumulation is caused by facilitated diffusion, or shuttling, of the Dorsal/Cactus complex. We also show that active Toll receptors are limiting in wild-type embryos, which is a key factor in explaining global Dorsal gradient formation. Our results suggest that shuttling is necessary for viability of embryos from mothers with compromised dorsal levels. Therefore, Cactus not only has the primary role of regulating Dorsal nuclear import, but also has a secondary role in shuttling. Given that this mechanism has been found in other, independent, systems, we suggest that it might be more prevalent than previously thought.

The sandfly Lutzomyia longipalpis LL5 embryonic cell line has active Toll and Imd pathways and shows immune responses to bacteria, yeast and Leishmania.

BackgroundLutzomyia longipalpis is the main vector of visceral leishmaniasis in Latin America. Sandfly immune responses are poorly understood. In previous work we showed that these vector insects respond to bacterial infections by modulating a defensin gene expression and activate the Imd pathway in response to Leishmania infection. Aspects of innate immune pathways in insects (including mosquito vectors of human diseases) have been revealed by studying insect cell lines, and we have previously demonstrated antiviral responses in the L. longipalpis embryonic cell line LL5.MethodsThe expression patterns of antimicrobial peptides (AMPs) and transcription factors were evaluated after silencing the repressors of the Toll pathway (cactus) and Imd pathway (caspar). AMPs and transcription factor expression patterns were also evaluated after challenge with heat-killed bacteria, heat-killed yeast, or live Leishmania.ResultsThese studies showed that LL5 cells have active Toll and Imd pathways, since they displayed an increased expression of AMP genes following silencing of the repressors cactus and caspar, respectively. These pathways were also activated by challenges with bacteria, yeast and Leishmania infantum chagasi.ConclusionsWe demonstrated that L. longipalpis LL5 embryonic cells respond to immune stimuli and are therefore a good model to study the immunological pathways of this important vector of leishmaniasis.

Nuclear envelope and chromatin, lock and key of genome integrity.

More than as an inert separation between the inside and outside of the nucleus, the nuclear envelope (NE) constitutes an active toll, which controls the import and export of molecules, and also a hub for a diversity of genomic processes, such as transcription, DNA repair, and chromatin dynamics. Proteins localized at the inner surface of the NE (such as lamins, nuclear pore proteins, lamin-associated proteins) interact with chromatin in a dynamic manner, contributing to the establishment of topological domains. In this review, we address the complex interplay between chromatin and NE. We discuss the divergence of this cross talk during evolution and comment both on the current established models and the most recent findings. In particular, we focus our attention on how the NE cooperates with chromatin in protecting the genome integrity.

No requirement for localized Nudel protein expression in Drosophila embryonic axis determination

Drosophila embryonic dorsal-ventral polarity is defined by a maternally encoded signal transduction pathway. Gastrulation Defective, Snake, and Easter comprise a serine protease cascade that operates in the perivitelline space to generate active ligand for the Toll receptor, which resides in the embryonic membrane. Toll is activated only on the ventral side of the embryo. Spatial regulation of this pathway is initiated by the ventrally restricted expression of the sulfotransferase Pipe in the follicular epithelium that surrounds the developing oocyte. Pipe is thought to modify a target molecule that is secreted and localized within the ventral region of the egg and future embryo, where it influences the activity of the pathway such that active Toll ligand is produced only ventrally. A potential substrate for Pipe is encoded by nudel, which is expressed throughout the follicle cell layer and encodes a large, multi-functional secreted protein that contains a serine protease domain as well as other structural features characteristic of extracellular matrix proteins. A previous mosaic analysis suggested that the protease domain of Nudel is not a target for Pipe activity as its expression is not required in pipe-expressing cells, but failed to rule out such a role for other functional domains of the protein. To investigate this possibility, we carried out a mosaic analysis of additional nudel alleles, including some that affect the entire protein. Our analysis demonstrated that proteolytically processed segments of Nudel are secreted into the perivitelline space and stably localized, as would be expected for the target of Pipe, However, we found no requirement for nudel to be expressed in ventral, pipe-expressing follicle cells, thereby eliminating Nudel as an essential substrate of Pipe sulfotransferase activity.

Role of the Spätzle Pro-domain in the Generation of an Active Toll Receptor Ligand

The cytokine Spätzle is the ligand for Drosophila Toll, the prototype of an important family of membrane receptors that function in embryonic patterning and innate immunity. A dimeric precursor of Spätzle is processed by an endoprotease to produce a form (C-106) that cross-links Toll receptor ectodomains and establishes signaling. Here we show that before processing the pro-domain of Spätzle is required for correct biosynthesis and secretion. We mapped two loss-of-function mutations of Spätzle to a discrete site in the pro-domain and showed that the phenotype arises because of a defect in biosynthesis rather than signaling. We also report that the pro-domain and C-106 remain associated after cleavage and that this processed complex signals with the same characteristics as the C-terminal fragment. These results suggest that before activation the determinants on C-106 that bind specifically to Toll are sequestered by the pro-domain and that proteolytic processing causes conformational rearrangements that expose these determinants and enables binding to Toll. Furthermore, we show that the pro-domain is released when the Toll extracellular domain binds to the complex, a finding that has implications for the generation of a signaling-competent Toll dimer.

The Drosophila melanogaster toll pathway participates in resistance to infection by the gram-negative human pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a gram-negative pathogen that infects immunocompromised and cystic fibrosis patients. The molecular basis of the host-P. aeruginosa interaction and the effect of specific P. aeruginosa virulence factors on various components of the innate immunity pathways are largely unknown. We examine interactions between P. aeruginosa virulence factors and components of innate immunity response in the Drosophila melanogaster model system to reveal the importance of the Toll signaling pathway in resistance to infection by the P. aeruginosa human isolate PA14. Using the two PA14-isogenic mutants plcS and dsbA, we show that Drosophila loss-of-function mutants of Spatzle, the extracellular ligand of Toll, and Dorsal and Dif, two NF-kappa B-like transcription factors, allow increased P. aeruginosa infectivity within fly tissues. In contrast, a constitutively active Toll mutant and a loss-of-function mutant of Cactus, an I kappa B-like factor that inhibits the Toll signaling, reduce infectivity. Our finding that Dorsal activity is required to restrict P. aeruginosa infectivity in Drosophila provides direct in vivo evidence for Dorsal function in adult fly immunity. Additionally, our results provide the basis for future studies into interactions between P. aeruginosa virulence factors and components of the Toll signaling pathway, which is functionally conserved between flies and humans.

Autophosphorylation of Pelle

Pelle is a downstream serine-threonine kinase in the Toll signaling pathway in Drosophila. Toll signaling is involved in both fly immune responses and in dorsal-ventral patterning. Shen and Manley investigated the mechanism by which Pelle is activated during Toll receptor stimulation. Pelle was phosphorylated in fly embryos at the time of Toll signaling, and flies with a gain-of-function Toll receptor exhibited enhanced Pelle phosphorylation. In transfected Schneider L2 cells, the phosphorylation status of Pelle was correlated with its ability to activate the downstream transcription factor Dorsal, and Pelle phosphorylation was enhanced by coexpression of the active mutant Toll receptor. In vitro experiments demonstrated that Pelle phosphorylation was essential for Pelle kinase activity. Autophosphorylating ability of Pelle was demonstrated by showing that phosphatase-treated Pelle preincubated with adenosine triphosphate yielded an activated Pelle. Phosphatase-treated Pelle could also phosphorylate a catalytically inactive Pelle mutant. Thus, the authors propose that Pelle exists in an inactive complex with the Toll receptor and that ligand binding induces dimerization of the complex, allowing Pelle to autophosphorylate, which then promotes the dissociation of Pelle from the complex for interaction with downstream substrates. B. Shen, J. L. Manley, Pelle kinase is activated by autophosphorylation during Toll signaling in Drosophila. Development 129, 1925-1933 (2002).[Online Journal]

Autophosphorylation of Pelle

Pelle is a downstream serine-threonine kinase in the Toll signaling pathway in Drosophila . Toll signaling is involved in both fly immune responses and in dorsal-ventral patterning. Shen and Manley investigated the mechanism by which Pelle is activated during Toll receptor stimulation. Pelle was phosphorylated in fly embryos at the time of Toll signaling, and flies with a gain-of-function Toll receptor exhibited enhanced Pelle phosphorylation. In transfected Schneider L2 cells, the phosphorylation status of Pelle was correlated with its ability to activate the downstream transcription factor Dorsal, and Pelle phosphorylation was enhanced by coexpression of the active mutant Toll receptor. In vitro experiments demonstrated that Pelle phosphorylation was essential for Pelle kinase activity. Autophosphorylating ability of Pelle was demonstrated by showing that phosphatase-treated Pelle preincubated with adenosine triphosphate yielded an activated Pelle. Phosphatase-treated Pelle could also phosphorylate a catalytically inactive Pelle mutant. Thus, the authors propose that Pelle exists in an inactive complex with the Toll receptor and that ligand binding induces dimerization of the complex, allowing Pelle to autophosphorylate, which then promotes the dissociation of Pelle from the complex for interaction with downstream substrates. B. Shen, J. L. Manley, Pelle kinase is activated by autophosphorylation during Toll signaling in Drosophila . Development 129 , 1925-1933 (2002). [Online Journal]

Relocalization of Drosophila dorsal protein can be induced by a rise in cytoplasmic calcium concentration and the expression of constitutively active but not wild-type Toll receptors.

The generation of dorso-ventral polarity in Drosophila relies on the formation of a nuclear gradient of the rel/nuclear factor kappa B transcription factor dorsal in the pre-cellular syncitial embryo by a process of differential nuclear localization. It is thought that the gradient is formed by activation at ventral positions of the membrane receptor Toll that in turn causes the local dissociation of dorsal from the cytoplasmic anchor protein cactus. Although Toll is related in its cytoplasmic domain to the interleukin-1 receptor little is known about the signal transduction pathways that lead from Toll to the relocalization of dorsal. In this paper we have used immunofluorescence microscopy as a direct assay of dorsal protein nuclear localization in the Drosophila cell line Schneider 2. We find that increased cytoplasmic calcium concentration and the expression of constitutively active Toll receptors can induce the relocalization of dorsal. By contrast, we find that activation of endogenous protein kinase A and expression of wild-type Toll receptors, which activate zen-chloramphenicol acetyltransferase reporter genes in this system, have only a marginal effect on the cellular distribution of the dorsal protein. Treatment of cells with activators of protein kinase C and radical oxygen intermediates, both of which activate nuclear factor kappa B, also has little effect on dorsal protein localization. We propose that different threshold levels of dorsal activation can be established by distinctly regulated signal transduction pathways.