RESUMO
Cancer exerts pleiotropic, systemic effects on organisms, leading to health deterioration and eventually to organismal death. How cancer induces systemic effects on remote organs and the organism itself still remains elusive. Here we describe a role for NetrinB (NetB), a protein with a particularly well-characterized role as a tissue-level axon guidance cue, in mediating oncogenic stress-induced organismal, metabolic reprogramming as a systemic humoral factor. In Drosophila, Ras-induced dysplastic cells upregulate and secrete NetB. Inhibition of either NetB from the transformed tissue or its receptor in the fat body suppresses oncogenic stress-induced organismal death. NetB from the dysplastic tissue remotely suppresses carnitine biosynthesis in the fat body, which is critical for acetyl-CoA generation and systemic metabolism. Supplementation of carnitine or acetyl-CoA ameliorates organismal health under oncogenic stress. This is the first identification, to our knowledge, of a role for the Netrin molecule, which has been studied extensively for its role within tissues, in humorally mediating systemic effects of local oncogenic stress on remote organs and organismal metabolism.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Netrinas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Acetilcoenzima A/metabolismo , Transdução de Sinais , Axônios/metabolismo , Fatores de Crescimento Neural/metabolismoRESUMO
Cells need to sense stresses to initiate the execution of the dormant cell death program. Since the discovery of the first BH3-only protein Bad, BH3-only proteins have been recognized as indispensable stress sensors that induce apoptosis. BH3-only proteins have so far not been identified in Drosophila despite their importance in other organisms. Here, we identify the first Drosophila BH3-only protein and name it sayonara. Sayonara induces apoptosis in a BH3 motif-dependent manner and interacts genetically and biochemically with the BCL-2 homologous proteins, Buffy and Debcl. There is a positive feedback loop between Sayonara-mediated caspase activation and autophagy. The BH3 motif of sayonara phylogenetically appeared at the time of the ancestral gene duplication that led to the formation of Buffy and Debcl in the dipteran lineage. To our knowledge, this is the first identification of a bona fide BH3-only protein in Drosophila, thus providing a unique example of how cell death mechanisms can evolve both through time and across taxa.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/genética , Apoptose/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas de Drosophila/metabolismoRESUMO
Sugar metabolism plays a pivotal role in sustaining life. Its dynamics within organisms is less understood compared to its intracellular metabolism. Galactose, a hexose stereoisomer of glucose, is a monosaccharide transported via the same transporters with glucose. Galactose feeds into glycolysis and regulates protein glycosylation. Defects in galactose metabolism are lethal for animals. Here, by transgenically implementing the yeast galactose sensing system into Drosophila, we developed a genetically encoded sensor, GALDAR, which detects galactose in vivo. Using this heterologous system, we revealed dynamics of galactose metabolism in various tissues. Notably, we discovered that intestinal stem cells do not uptake detectable levels of galactose or glucose. GALDAR elucidates the role for galactokinase in metabolism of galactose and a transition of galactose metabolism during the larval period. This work provides a new system that enables analyses of in vivo sugar metabolism.
Assuntos
Galactose , Glicólise , Animais , Galactose/metabolismo , Glicólise/genética , Glicosilação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Drosophila/metabolismo , Glucose/metabolismoRESUMO
Many adult tissues are composed of differentiated cells and stem cells, each working in a coordinated manner to maintain tissue homeostasis during physiological cell turnover. Old differentiated cells are believed to typically die by apoptosis. Here, we discovered a previously uncharacterized, new phenomenon, which we name erebosis based on the ancient Greek word erebos ("complete darkness"), in the gut enterocytes of adult Drosophila. Cells that undergo erebosis lose cytoskeleton, cell adhesion, organelles and fluorescent proteins, but accumulate Angiotensin-converting enzyme (Ance). Their nuclei become flat and occasionally difficult to detect. Erebotic cells do not have characteristic features of apoptosis, necrosis, or autophagic cell death. Inhibition of apoptosis prevents neither the gut cell turnover nor erebosis. We hypothesize that erebosis is a cell death mechanism for the enterocyte flux to mediate tissue homeostasis in the gut.
Assuntos
Drosophila , Enterócitos , Animais , Apoptose , Morte Celular , Drosophila/metabolismo , Enterócitos/metabolismo , HomeostaseRESUMO
Tissue wounding induces the rapid recruitment of leukocytes. Wounds and tumours--a type of 'unhealed wound'--generate hydrogen peroxide (H(2)O(2)) through an NADPH oxidase (NOX). This extracellular H(2)O(2) mediates recruitment of leukocytes, particularly the first responders of innate immunity, neutrophils, to injured tissue. However, the sensor that neutrophils use to detect the redox state at wounds is unknown. Here we identify the Src family kinase (SFK) Lyn as a redox sensor that mediates initial neutrophil recruitment to wounds in zebrafish larvae. Lyn activation in neutrophils is dependent on wound-derived H(2)O(2) after tissue injury, and inhibition of Lyn attenuates neutrophil wound recruitment. Inhibition of SFKs also disrupted H(2)O(2)-mediated chemotaxis of primary human neutrophils. In vitro analysis identified a single cysteine residue, C466, as being responsible for direct oxidation-mediated activation of Lyn. Furthermore, transgenic-tissue-specific reconstitution with wild-type Lyn and a cysteine mutant revealed that Lyn C466 is important for the neutrophil wound response and downstream signalling in vivo. This is the first identification, to our knowledge, of a physiological redox sensor that mediates leukocyte wound attraction in multicellular organisms.
Assuntos
Neutrófilos/enzimologia , Oxirredução , Ferimentos e Lesões/enzimologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Quinases da Família src/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Larva , Peixe-Zebra/metabolismoRESUMO
Neutrophil recruitment to sites of injury or infection is essential for host defense, but it needs to be tightly regulated to prevent tissue damage. Phosphoinositide 3-kinase (PI3K), which generates the phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P(3)], is necessary for neutrophil motility in vivo; however, the role of SH2-domain-containing 5-inositol phosphatase (SHIP) enzymes, which hydrolyze PI(3,4,5)P(3) to phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)], is not well understood. Here we show that SHIP phosphatases limit neutrophil motility in live zebrafish. Using real-time imaging of bioprobes specific for PI(3,4,5)P(3) and PI(3,4)P(2) in neutrophils, we found that PI(3,4,5)P(3) and PI(3,4)P(2) accumulate at the leading edge while PI(3,4)P(2) also localizes to the trailing edge of migrating neutrophils in vivo. Depletion of SHIP phosphatases using morpholino oligonucleotides led to increased neutrophil 3D motility and neutrophil infiltration into wounds. The increase in neutrophil wound recruitment in SHIP morphants was rescued by treatment with low dose PI3Kγ inhibitor, suggesting that SHIP limits neutrophil motility by modulating PI3K signaling. Moreover, overexpression of the SHIP phosphatase domain in neutrophils impaired neutrophil 3D migration. Taken together, our findings suggest that SHIP phosphatases control neutrophil inflammation by limiting neutrophil motility in vivo.
Assuntos
Neutrófilos/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Cicatrização , Proteínas de Peixe-Zebra/metabolismo , Animais , Movimento Celular , Embrião não Mamífero/citologia , Embrião não Mamífero/enzimologia , Embrião não Mamífero/imunologia , Expressão Gênica , Neutrófilos/imunologia , Neutrófilos/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Transporte Proteico , Sistemas do Segundo Mensageiro , Cauda , Imagem com Lapso de Tempo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Microtubules control cell motility by positively regulating polarization in many cell types. However, how microtubules regulate leukocyte migration is not well understood, particularly in living organisms. Here we exploited the zebrafish system to study the role of microtubules in neutrophil migration in vivo. The localization of microtubules was visualized in motile neutrophils using various bioprobes, revealing that, in contrast to what has been seen in studies in vitro, the microtubule organizing center is positioned in front of the nucleus (relative to the direction of migration) in motile neutrophils. Microtubule disassembly impaired attraction of neutrophils to wounds but enhanced the polarity of F-actin dynamics as measured by the distribution of stable and dynamic F-actin. Microtubule depolymerization inhibited polarized phosphoinositol 3-kinase (PI(3)K) activation at the leading edge and induced rapid PI(3)K independent motility. Finally, we show that microtubules exert their effects on neutrophil polarity and motility at least in part by the negative regulation of both Rho and Rac activity. These results provide new insight into the role of microtubules in neutrophil migration in a living vertebrate and show that the motility of these professional migratory cells are subject to distinctly different rules from those established for other cell types.
Assuntos
Microtúbulos/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Peixe-Zebra/metabolismo , Animais , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Quimiotaxia/fisiologiaRESUMO
Cell death and proliferation are at a glance dichotomic events, but occasionally coupled. Caspases, traditionally known to execute apoptosis, play non-apoptotic roles, but their exact mechanism remains elusive. Here, using Drosophila intestinal stem cells (ISCs), we discovered that activation of caspases induces massive cell proliferation rather than cell death. We elucidate that a positive feedback circuit exists between caspases and JNK, which can simultaneously drive cell proliferation and cell death. In ISCs, signalling from JNK to caspases is defective, which skews the balance towards proliferation. Mechanistically, two-tiered regulation of the DIAP1 inhibitor rpr, through its transcription and its protein localization, exists. This work provides a conceptual framework that explains how caspases perform apoptotic and non-apoptotic functions in vivo and how ISCs accomplish their resistance to cell death.
Assuntos
Proteínas de Drosophila , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Retroalimentação , Proteínas Inibidoras de Apoptose/metabolismo , Morte Celular , Drosophila/metabolismo , Caspases/metabolismo , Proliferação de Células/genética , Células-Tronco/metabolismoRESUMO
Historically, necrosis has been considered a passive process, which is induced by extreme stress or damage. However, recent findings of necroptosis, a programmed form of necrosis, shed a new light on necrosis. It has been challenging to detect necrosis reliably in vivo, partly due to the lack of genetically encoded sensors to detect necrosis. This is in stark contrast with the availability of many genetically encoded biosensors for apoptosis. Here we developed Necrosensor, a genetically encoded fluorescent sensor that detects necrosis in Drosophila, by utilizing HMGB1, which is released from the nucleus as a damage-associated molecular pattern (DAMP). We demonstrate that Necrosensor is able to detect necrosis induced by various stresses in multiple tissues in both live and fixed conditions. Necrosensor also detects physiological necrosis that occurs during spermatogenesis in the testis. Using Necrosensor, we discovered previously unidentified, physiological necrosis of hemocyte progenitors in the hematopoietic lymph gland of developing larvae. This work provides a new transgenic system that enables in vivo detection of necrosis in real time without any intervention.
Assuntos
Técnicas Biossensoriais , Drosophila , Masculino , Animais , Drosophila/genética , Necrose , Apoptose , EspermatogêneseRESUMO
CXCR4 is a G protein-coupled chemokine receptor that has been implicated in the pathogenesis of primary immunodeficiency disorders and cancer. Autosomal dominant gain-of-function truncations of CXCR4 are associated with warts, hypo-gammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a primary immunodeficiency disorder characterized by neutropenia and recurrent infections. Recent progress has implicated CXCR4-SDF1 (stromal cell-derived factor 1) signaling in regulating neutrophil homeostasis, but the precise role of CXCR4-SDF1 interactions in regulating neutrophil motility in vivo is not known. Here, we use the optical transparency of zebrafish to visualize neutrophil trafficking in vivo in a zebrafish model of WHIM syndrome. We demonstrate that expression of WHIM mutations in zebrafish neutrophils induces neutrophil retention in hematopoietic tissue, impairing neutrophil motility and wound recruitment. The neutrophil retention signal induced by WHIM truncation mutations is SDF1 dependent, because depletion of SDF1 with the use of morpholino oligonucleotides restores neutrophil chemotaxis to wounds. Moreover, localized activation of a genetically encoded, photoactivatable Rac guanosine triphosphatase is sufficient to direct migration of neutrophils that express the WHIM mutation. The findings suggest that this transgenic zebrafish model of WHIM syndrome may provide a valuable tool to screen for agents that modify CXCR4-SDF1 retention signals.
Assuntos
Neutropenia/genética , Neutropenia/patologia , Neutrófilos/fisiologia , Agamaglobulinemia/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Movimento Celular , Quimiocina CXCL12/genética , Quimiotaxia de Leucócito , Modelos Animais de Doenças , Expressão Gênica , Hematopoese , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/patologia , Dados de Sequência Molecular , Mutação , Neutrófilos/patologia , Receptores CXCR4/genética , Transdução de Sinais , Síndrome , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
The receptor tyrosine kinase Ror2 plays important roles in developmental morphogenesis. It has recently been shown that Ror2 mediates Wnt5a-induced noncanonical Wnt signaling by activating the Wnt-JNK pathway and inhibiting the beta-catenin-TCF pathway. However, the function of Ror2 in noncanonical Wnt signaling leading to cell migration is largely unknown. We show, using genetically different or manipulated cultured cells, that Ror2 is critical for Wnt5a-induced, but not Wnt3a-induced, cell migration. Ror2-mediated cell migration requires the extracellular cysteine-rich domain (CRD), which is the binding site for Wnt5a, and the cytoplasmic proline-rich domain (PRD) of Ror2. Furthermore, Ror2 can mediate filopodia formation via actin reorganization, irrespective of Wnt5a, and this Ror2-mediated filopodia formation requires the actin-binding protein filamin A, which associates with the PRD of Ror2. Intriguingly, disruption of filopodia formation by suppressing the expression of either Ror2 or filamin A inhibits Wnt5a-induced cell migration, indicating that Ror2-mediated filopodia formation is essential for Wnt5a-induced cell migration.
Assuntos
Movimento Celular/efeitos dos fármacos , Proteínas Proto-Oncogênicas/farmacologia , Pseudópodes/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Wnt/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Contráteis/química , Meios de Cultivo Condicionados , Citoplasma/efeitos dos fármacos , Proteínas Desgrenhadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Filaminas , Células HeLa , Humanos , Camundongos , Proteínas dos Microfilamentos/química , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/química , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase , Proteína Wnt-5aRESUMO
Tissue integrity is contingent on maintaining stem cells. Intestinal stem cells (ISCs) over-proliferate during ageing, leading to tissue dysplasia in Drosophila melanogaster. Here we describe a role for white, encoding the evolutionarily conserved ATP-binding cassette transporter subfamily G, with a particularly well-characterized role in eye colour pigmentation, in ageing-induced ISC proliferation in the midgut. ISCs increase expression of white during ageing. ISC-specific inhibition of white suppresses ageing-induced ISC dysregulation and prolongs lifespan. Of the proteins that form heterodimers with White, Brown mediates ISC dysregulation during ageing. Metabolomics analyses reveal previously unappreciated, profound metabolic impacts of white inhibition on organismal metabolism. Among the metabolites affected by White, tetrahydrofolate is transported by White, is accumulated in ISCs during ageing and is indispensable for ageing-induced ISC over-proliferation. Since Thomas Morgan's isolation of a white mutant as the first Drosophila mutant, white mutants have been used extensively as genetic systems and often as controls. Our findings provide insights into metabolic regulation of stem cells mediated by the classic gene white.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/fisiologia , Envelhecimento/genética , Envelhecimento/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Proteínas do Olho/genética , Proteínas do Olho/fisiologia , Homeostase/genética , Homeostase/fisiologia , Intestinos/fisiologia , Células-Tronco/fisiologia , Animais , Proliferação de Células , Drosophila melanogaster/genética , Cor de Olho/genética , Ácido Fólico/metabolismo , Intestinos/citologia , Intestinos/crescimento & desenvolvimento , MetabolômicaRESUMO
Oncogenes often promote cell death as well as proliferation. How oncogenes drive these diametrically opposed phenomena remains to be solved. A key question is whether cell death occurs as a response to aberrant proliferation signals or through a proliferation-independent mechanism. Here, we reveal that Src, the first identified oncogene, simultaneously drives cell proliferation and death in an obligatorily coupled manner through parallel MAPK pathways. The two MAPK pathways diverge from a lynchpin protein Slpr. A MAPK p38 drives proliferation whereas another MAPK JNK drives apoptosis independently of proliferation signals. Src-p38-induced proliferation is regulated by methionine-mediated Tor signaling. Reduction of dietary methionine uncouples the obligatory coupling of cell proliferation and death, suppressing tumorigenesis and tumor-induced lethality. Our findings provide an insight into how cells evolved to have a fail-safe mechanism that thwarts tumorigenesis by the oncogene Src. We also exemplify a diet-based approach to circumvent oncogenesis by exploiting the fail-safe mechanism.
Assuntos
Morte Celular , Proliferação de Células , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Metionina/deficiência , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismoRESUMO
The maintenance of genomic stability is an essential cellular function for a variety of well-coordinated regulation of biological activities of organisms, and a failure in its function results in the accumulation of mutations and/or abnormality in the induction of apoptosis, eventually leading to onsets of various diseases, including malignant tumors. DNA damage responses, in particular cell-cycle checkpoint regulation, play important roles in maintaining genomic integrity. In response to DNA damages induced by gamma-irradiation, ultraviolet irradiation, various chemicals, or reactive oxygen species (ROS), intrinsic cell-cycle checkpoint machinery is rapidly activated to arrest cells at particular cell-cycle points, and during cell-cycle checkpoint arrest cells may try to repair damaged DNAs, and then re-start cell-cycle upon the completion of DNA repair. Alternatively, if the extents of DNA damage overwhelm the capacity of the cellular repair machinery, cells may undergo apoptosis to prevent the accumulation of mutations within the organisms. In this article, we will first explain about our current view of DNA damage responses, in particular cell-cycle checkpoint regulation, and summarize our knowledge of the relationships between abnormalities of genes involved in DNA damage responses and malignant tumors, including hematopoietic malignancies. We will also discuss a possible implication of DNA damage responses in autoimmune diseases, such as rheumatoid arthritis.
Assuntos
Doenças Autoimunes/genética , Dano ao DNA , Genes cdc , Apoptose , Ataxia Telangiectasia/genética , Doenças Autoimunes/patologia , Ciclo Celular , Doença de Hodgkin/genética , Humanos , Linfoma/genética , MutaçãoRESUMO
In most multicellular organisms, homeostasis is contingent upon maintaining epithelial integrity. When unanticipated insults breach epithelial barriers, dormant programmes of tissue repair are immediately activated. However, many of the mechanisms that repair damaged epithelia remain poorly characterized. Here we describe a role for Plexin A (PlexA), a protein with particularly well-characterized roles in axonal pathfinding, in the healing of damaged epithelia in Drosophila. Semaphorins, which are PlexA ligands, also regulate tissue repair. We show that Drosophila PlexA has GAP activity for the Rap1 GTPase, which is known to regulate the stability of adherens junctions. Our observations suggest that the inhibition of Rap1 activity by PlexA in damaged Drosophila epithelia allows epithelial remodelling, thus facilitating wound repair. We also demonstrate a role for Plexin A1, a zebrafish orthologue of Drosophila PlexA, in epithelial repair in zebrafish tail fins. Thus, plexins function in epithelial wound healing in diverse taxa.
Assuntos
Moléculas de Adesão Celular/metabolismo , Drosophila melanogaster/metabolismo , Epitélio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Cicatrização , Peixe-Zebra/metabolismo , Actinas/metabolismo , Junções Aderentes/metabolismo , Nadadeiras de Animais/metabolismo , Animais , Células Epiteliais/metabolismo , Interferência de RNA , Transdução de SinaisRESUMO
Tissue injury can lead to scar formation or tissue regeneration. How regenerative animals sense initial tissue injury and transform wound signals into regenerative growth is an unresolved question. Previously, we found that the Src family kinase (SFK) Lyn functions as a redox sensor in leukocytes that detects H(2)O(2) at wounds in zebrafish larvae. In this paper, using zebrafish larval tail fins as a model, we find that wounding rapidly activated SFK and calcium signaling in epithelia. The immediate SFK and calcium signaling in epithelia was important for late epimorphic regeneration of amputated fins. Wound-induced activation of SFKs in epithelia was dependent on injury-generated H(2)O(2). A SFK member, Fynb, was responsible for fin regeneration. This work provides a new link between early wound responses and late regeneration and suggests that redox, SFK, and calcium signaling are immediate "wound signals" that integrate early wound responses and late epimorphic regeneration.
Assuntos
Sinalização do Cálcio , Regeneração/fisiologia , Cicatrização/fisiologia , Quinases da Família src/metabolismo , Nadadeiras de Animais/lesões , Nadadeiras de Animais/metabolismo , Animais , Peróxido de Hidrogênio/metabolismo , Oxirredução , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Proto-Oncogênicas c-yes/genética , Proteínas Proto-Oncogênicas c-yes/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
How neutrophils traffic during inflammation in vivo remains elusive. To visualize the origin and fate of neutrophils during induction and resolution of inflammation, we established a genetically encoded photolabeling system by generating transgenic zebrafish that express a photoconvertible fluorescent reporter Dendra2 in neutrophils. Spatiotemporal photolabeling of neutrophils in vivo demonstrates that they emerge from the hematopoietic tissue in close proximity to injured tissue and repeat forward and reverse migration between the wound and the vasculature. Subsequently, neutrophils disperse throughout the body as wound-healing proceeds, contributing to local resolution at injured tissue and systemic dissemination of wound-sensitized neutrophils. Tissue damage also alters the fate of neutrophils in the caudal hematopoietic tissue and promotes caudorostral mobilization of neutrophils via the circulation to the cephalic mesenchyme. This work provides new insight into neutrophil behaviors during inflammation and resolution within a multicellular organism.
Assuntos
Movimento Celular , Modelos Animais de Doenças , Inflamação/patologia , Luz , Proteínas Luminescentes/química , Neutrófilos/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Embrião não Mamífero/metabolismo , Embrião não Mamífero/efeitos da radiação , Processamento de Imagem Assistida por Computador , Inflamação/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Neutrófilos/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cicatrização , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Neutrophil homeostasis is essential for host defense. Here we identify dual roles for Rac2 during neutrophil homeostasis using a zebrafish model of primary immune deficiency induced by the human inhibitory Rac2D57N mutation in neutrophils. Noninvasive live imaging of Rac2 morphants or Rac2D57N zebrafish larvae demonstrates an essential role for Rac2 in regulating 3D motility and the polarization of F-actin dynamics and PI(3)K signaling in vivo. Tracking of photolabeled Rac2-deficient neutrophils from hematopoietic tissue also shows increased mobilization into the circulation, indicating that neutrophil mobilization does not require traditionally defined cell motility. Moreover, excessive neutrophil retention in hematopoietic tissue resulting from a constitutively active CXCR4 mutation in zebrafish warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is partially rescued by the inhibitory Rac2 mutation. These findings reveal that Rac2 signaling is necessary for both neutrophil 3D motility and CXCR4-mediated neutrophil retention in hematopoietic tissue, thereby limiting neutrophil mobilization, a critical first step in the innate immune response.
Assuntos
Movimento Celular/fisiologia , Sistema Hematopoético/fisiologia , Neutrófilos/citologia , Peixe-Zebra/genética , Proteínas rac de Ligação ao GTP/fisiologia , Actinas/metabolismo , Agamaglobulinemia/complicações , Animais , Animais Geneticamente Modificados , Bactérias , Infecções Bacterianas/complicações , Western Blotting , Medula Óssea/metabolismo , Imunofluorescência , Células HL-60 , Humanos , Síndromes de Imunodeficiência/complicações , Larva/metabolismo , Larva/microbiologia , Mutação/genética , Neutrófilos/metabolismo , RNA Mensageiro/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Verrugas/complicações , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteína RAC2 de Ligação ao GTPRESUMO
Cell polarity is crucial for directed migration. Here we show that phosphoinositide 3-kinase (PI(3)K) mediates neutrophil migration in vivo by differentially regulating cell protrusion and polarity. The dynamics of PI(3)K products PI(3,4,5)P(3)-PI(3,4)P(2) during neutrophil migration were visualized in living zebrafish, revealing that PI(3)K activation at the leading edge is critical for neutrophil motility in intact tissues. A genetically encoded photoactivatable Rac was used to demonstrate that localized activation of Rac is sufficient to direct migration with precise temporal and spatial control in vivo. Similar stimulation of PI(3)K-inhibited cells did not direct migration. Localized Rac activation rescued membrane protrusion but not anteroposterior polarization of F-actin dynamics of PI(3)K-inhibited cells. Uncoupling Rac-mediated protrusion and polarization suggests a paradigm of two-tiered PI(3)K-mediated regulation of cell motility. This work provides new insight into how cell signaling at the front and back of the cell is coordinated during polarized cell migration in intact tissues within a multicellular organism.