RESUMEN
Glia are essential to protecting and enabling nervous system function and a key glial function is the formation of the glial sheath around peripheral axons. Each peripheral nerve in the Drosophila larva is ensheathed by three glial layers, which structurally support and insulate the peripheral axons. How peripheral glia communicate with each other and between layers is not well established and we investigated the role of Innexins in mediating glial function in the Drosophila periphery. Of the eight Drosophila Innexins, we found two (Inx1 and Inx2) are important for peripheral glia development. In particular loss of Inx1 and Inx2 resulted in defects in the wrapping glia leading to disruption of the glia wrap. Of interest loss of Inx2 in the subperineurial glia also resulted in defects in the neighboring wrapping glia. Inx plaques were observed between the subperineurial glia and the wrapping glia suggesting that gap junctions link these two glial cell types. We found Inx2 is key to Ca2+ pulses in the peripheral subperineurial glia but not in the wrapping glia, and we found no evidence of gap junction communication between subperineurial and wrapping glia. Rather we have clear evidence that Inx2 plays an adhesive and channel-independent role between the subperineurial and wrapping glia to ensure the integrity of the glial wrap.SIGNIFICANCE STATEMENT Gap junctions are critical for glia communication and formation of myelin in myelinating glia. However, the role of gap junctions in non-myelinating glia is not well studied, yet non-myelinating glia are critical for peripheral nerve function. We found the Innexin gap junction proteins are present between different classes of peripheral glia in Drosophila. Here Innexins form junctions to facilitate adhesion between the different glia but do so in a channel-independent manner. Loss of adhesion leads to disruption of the glial wrap around axons and leads to fragmentation of the wrapping glia membranes. Our work points to an important role for gap junction proteins in mediating insulation by non-myelinating glia.
Asunto(s)
Proteínas de Drosophila , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Neuroglía/metabolismo , Sistema Nervioso Periférico/metabolismo , Drosophila/metabolismo , Axones/metabolismo , Conexinas/genética , Conexinas/metabolismoRESUMEN
Junctional complexes that mediate cell adhesion are key to epithelial integrity, cell division and permeability barrier formation. In Drosophila, the scaffolding proteins Scribble (Scrib) and Discs Large (Dlg) are key regulators of epithelial polarity, proliferation, assembly of junctions and protein trafficking. We found that Scrib and Dlg are necessary for the formation of the tricellular junction (TCJ), a unique junction that forms in epithelia at the point of convergence of three neighboring cells. Scrib and Dlg are in close proximity with the TCJ proteins Gliotactin (Gli) and Bark Beetle (Bark), and both are required for TCJ protein recruitment. Loss of Bark or Gli led to basolateral spread of the TCJ complex at the cell corners. Loss of the septate junction proteins Nrx-IV and the Na+/K+ ATPase also resulted in basolateral spread of the entire TCJ complex at the cell corners. The Scrib PDZ1-2 domains and the Dlg GUK domain are necessary for Bark and Gli localization to the TCJ. Overall, we propose a model in which Scrib and Dlg are key components of the TCJ, and form a complex with Bark and Gli.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Uniones Intercelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas de Drosophila/química , Técnicas de Silenciamiento del Gen , Proteínas de la Membrana/química , Dominios ProteicosRESUMEN
The Drosophila nervous system is ensheathed by a layer of outer glial cells, the perineurial glia, and a specialized extracellular matrix, the neural lamella. The function of perineurial glial cells and how they interact with the extracellular matrix are just beginning to be elucidated. Integrin-based focal adhesion complexes link the glial membrane to the extracellular matrix, but little is known about integrin's regulators in the glia. The transmembrane Ig domain protein Basigin/CD147/EMMPRIN is highly expressed in the perineurial glia surrounding the Drosophila larval nervous system. Here we show that Basigin associates with integrin at the focal adhesions to uphold the structure of the glia-extracellular matrix sheath. Knockdown of Basigin in perineurial glia using RNAi results in significant shortening of the ventral nerve cord, compression of the glia and extracellular matrix in the peripheral nerves, and reduction in larval locomotion. We determined that Basigin is expressed in close proximity to integrin at the glial membrane, and that expression of the extracellular integrin-binding domain of Basigin is sufficient to rescue peripheral glial compression. We also found that a reduction in expression of integrin at the membrane rescues the ventral nerve cord shortening, peripheral glial compression, and locomotor phenotypes, and that reduction in the integrin-binding protein Talin can partially rescue glial compression. These results identify Basigin as a potential negative regulator of integrin in the glia, supporting proper glial and extracellular matrix ensheathment of the nervous system.SIGNIFICANCE STATEMENT The glial cells and extracellular matrix play important roles in supporting and protecting the nervous system, but the interactions between these components have not been well characterized. Our study identified expression of a conserved Ig superfamily protein, Basigin, at the glial membrane of Drosophila where it associates with the integrin-based focal adhesion complexes to ensure proper ensheathment of the CNS and PNS. Loss of Basigin in the glia results in an overall compression of the nervous system due to integrin dysregulation, which causes locomotor defects in the animals. This underlies the importance of glia-matrix communication for structural and functional support of the nervous system.
Asunto(s)
Proteínas de Drosophila/metabolismo , Integrinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Neuroglía/metabolismo , Nervios Periféricos/metabolismo , Animales , Adhesión Celular/fisiología , Drosophila melanogaster , Matriz Extracelular/metabolismo , Larva/metabolismo , Locomoción/fisiología , Neuroglía/citología , Nervios Periféricos/citología , Interferencia de ARNRESUMEN
Epithelial junctions ensure cell-cell adhesion and establish permeability barriers between cells. At the corners of epithelia, the tricellular junction (TCJ) is formed by three adjacent epithelial cells and generates a functional barrier. In Drosophila, a key TCJ protein is Gliotactin (Gli) where loss of Gli disrupts barrier formation and function. Conversely, overexpressed Gli spreads away from the TCJ and triggers apoptosis, delamination, and cell migration. Thus, Gli protein levels are tightly regulated and by two mechanisms, at the protein levels by tyrosine phosphorylation and endocytosis and at the mRNA level through microRNA-184. Regulation of Gli mRNA is mediated through a Gli-BMP-miR184 feedback loop. Excessive Gli triggers BMP signaling pathway through the activation of Tkv type-I BMP receptor and Mad. Elevated level of pMad induces micrRNA-184 expression which in turn targets the Gli 3'UTR and mRNA degradation. Gli activation of Tkv is not through its ligand Dpp but rather through the inhibition of Dad, an inhibitory-Smad. Here, we show that ectopic expression of Gli interferes with Tkv-Dad association by sequestering Dad away from Tkv. The reduced inhibitory effect of Dad on Tkv results in the increased Tkv-pMad signaling activity, and this effect is continuous through larval and pupal wing formation.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila , Proteínas de la Membrana , MicroARNs , Proteínas del Tejido Nervioso , Transducción de Señal , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , MicroARNs/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas , ARN Mensajero , Receptores de Superficie CelularRESUMEN
Epithelial bicellular and tricellular junctions are essential for establishing and maintaining permeability barriers. Tricellular junctions are formed by the convergence of three bicellular junctions at the corners of neighbouring epithelia. Gliotactin, a member of the Neuroligin family, is located at theDrosophilatricellular junction, and is crucial for the formation of tricellular and septate junctions, as well as permeability barrier function. Gliotactin protein levels are tightly controlled by phosphorylation at tyrosine residues and endocytosis. Blocking endocytosis or overexpressing Gliotactin results in the spread of Gliotactin from the tricellular junction, resulting in apoptosis, delamination and migration of epithelial cells. We show that Gliotactin levels are also regulated at the mRNA level by micro (mi)RNA-mediated degradation and that miRNAs are targeted to a short region in the 3'UTR that includes a conserved miR-184 target site. miR-184 also targets a suite of septate junction proteins, including NrxIV, coracle and Mcr. miR-184 expression is triggered when Gliotactin is overexpressed, leading to activation of the BMP signalling pathway. Gliotactin specifically interferes with Dad, an inhibitory SMAD, leading to activation of the Tkv type-I receptor and activation of Mad to elevate the biogenesis and expression of miR-184.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas de la Membrana/metabolismo , MicroARNs/biosíntesis , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Apoptosis/fisiología , Movimiento Celular/fisiología , Citocinas/metabolismo , Proteínas de Drosophila/antagonistas & inhibidores , Endocitosis/fisiología , Activación Enzimática , Proteínas de la Membrana/genética , MicroARNs/genética , Proteínas del Tejido Nervioso/genética , Serpinas/metabolismo , Transducción de Señal/genética , Uniones Estrechas/fisiologíaRESUMEN
Cervical cancer is one of the leading causes of cancer death in women worldwide. The causative agents of cervical cancers, high-risk human papillomaviruses (HPVs), cause cancer through the action of two oncoproteins, E6 and E7. The E6 oncoprotein cooperates with an E3 ubiquitin ligase (UBE3A) to target the p53 tumour suppressor and important polarity and junctional PDZ proteins for proteasomal degradation, activities that are believed to contribute towards malignancy. However, the causative link between degradation of PDZ proteins and E6-mediated malignancy is largely unknown. We have developed an in vivo model of HPV E6-mediated cellular transformation using the genetic model organism, Drosophila melanogaster. Co-expression of E6 and human UBE3A in wing and eye epithelia results in severe morphological abnormalities. Furthermore, E6, via its PDZ-binding motif and in cooperation with UBE3A, targets a suite of PDZ proteins that are conserved in human and Drosophila, including Magi, Dlg and Scribble. Similar to human epithelia, Drosophila Magi is a major degradation target. Magi overexpression rescues the cellular abnormalities caused by E6+UBE3A coexpression and this activity of Magi is PDZ domain-dependent. Drosophila p53 was not targeted by E6+UBE3A, and E6+UBE3A activity alone is not sufficient to induce tumorigenesis, which only occurs when E6+UBE3A are expressed in conjunction with activated/oncogenic forms of Ras or Notch. Finally, through a genetic screen we have identified the insulin receptor signaling pathway as being required for E6+UBE3A induced hyperplasia. Our results suggest a highly conserved mechanism of HPV E6 mediated cellular transformation, and establish a powerful genetic model to identify and understand the cellular mechanisms that underlie HPV E6-induced malignancy.
Asunto(s)
Transformación Celular Viral/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Receptor de Insulina/metabolismo , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Drosophila melanogaster , Humanos , Inmunohistoquímica , Ubiquitina-Proteína LigasasRESUMEN
The nervous system is surrounded by an extracellular matrix composed of large glycoproteins, including perlecan, collagens, and laminins. Glial cells in many organisms secrete laminin, a large heterotrimeric protein consisting of an α, ß, and γ subunit. Prior studies have found that loss of laminin subunits from vertebrate Schwann cells causes loss of myelination and neuropathies, results attributed to loss of laminin-receptor signaling. We demonstrate that loss of the laminin γ subunit (LanB2) in the peripheral glia of Drosophila melanogaster results in the disruption of glial morphology due to disruption of laminin secretion. Specifically, knockdown of LanB2 in peripheral glia results in accumulation of the ß subunit (LanB1), leading to distended endoplasmic reticulum (ER), ER stress, and glial swelling. The physiological consequences of disruption of laminin secretion in glia included decreased larval locomotion and ultimately lethality. Loss of the γ subunit from wrapping glia resulted in a disruption in the glial ensheathment of axons but surprisingly did not affect animal locomotion. We found that Tango1, a protein thought to exclusively mediate collagen secretion, is also important for laminin secretion in glia via a collagen-independent mechanism. However loss of secretion of the laminin trimer does not disrupt animal locomotion. Rather, it is the loss of one subunit that leads to deleterious consequences through the accumulation of the remaining subunits. SIGNIFICANCE STATEMENT: This research presents a new perspective on how mutations in the extracellular matrix protein laminin cause severe consequences in glial wrapping and function. Glial-specific loss of the ß or γ laminin subunit disrupted glia morphology and led to ER expansion and stress due to retention of other subunits. The retention of the unpaired laminin subunit was key to the glial disruption as loss of Tango1 blocked secretion of the complete laminin trimer but did not lead to glial or locomotion defects. The effects were observed in the perineurial glia that envelope the peripheral and central nervous systems, providing evidence for the importance of this class of glia in supporting nervous system function.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Laminina/metabolismo , Larva/fisiología , Locomoción/fisiología , Sistema Nervioso/citología , Neuroglía/fisiología , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Colágeno/fisiología , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Laminina/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso/crecimiento & desarrollo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Many aspects of glial development are regulated by extracellular signals, including those from the extracellular matrix (ECM). Signals from the ECM are received by cell surface receptors, including the integrin family. Previously, we have shown that Drosophila integrins form adhesion complexes with Integrin-linked kinase and talin in the peripheral nerve glia and have conserved roles in glial sheath formation. However, integrin function in other aspects of glial development is unclear. The Drosophila eye imaginal disc (ED) and optic stalk (OS) complex is an excellent model with which to study glial migration, differentiation and glia-neuron interactions. We studied the roles of the integrin complexes in these glial developmental processes during OS/eye development. The common beta subunit ßPS and two alpha subunits, αPS2 and αPS3, are located in puncta at both glia-glia and glia-ECM interfaces. Depletion of ßPS integrin and talin by RNAi impaired the migration and distribution of glia within the OS resulting in morphological defects. Reduction of integrin or talin in the glia also disrupted photoreceptor axon outgrowth leading to axon stalling in the OS and ED. The neuronal defects were correlated with a disruption of the carpet glia tube paired with invasion of glia into the core of the OS and the formation of a glial cap. Our results suggest that integrin-mediated extracellular signals are important for multiple aspects of glial development and non-autonomously affect axonal migration during Drosophila eye development.
Asunto(s)
Axones/metabolismo , Drosophila melanogaster/fisiología , Adhesiones Focales/metabolismo , Neuroglía/citología , Visión Ocular/fisiología , Animales , Axones/fisiología , Adhesión Celular , Diferenciación Celular , Movimiento Celular , Drosophila melanogaster/embriología , Matriz Extracelular/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Discos Imaginales/citología , Integrinas/metabolismo , Neuronas/metabolismo , Fenotipo , Células Fotorreceptoras de Invertebrados/metabolismo , Interferencia de ARN , Talina/metabolismoRESUMEN
Establishment and maintenance of permeability barriers is one of the most important functions of epithelial cells. Tricellular junctions (TCJs) maintain the permeability barriers at the contact site of three epithelial cells. Gliotactin, a member of the Neuroligin family, is the only known Drosophila protein exclusively localized to the TCJ and is necessary for maintenance of the permeability barrier. Overexpression triggers the spread of Gliotactin away from the TCJ and causes epithelial cells to delaminate, migrate and die. Furthermore, excess Gliotactin at the cell membrane results in an extensive downregulation of Discs large (Dlg) at the septate junctions. The intracellular domain of Gliotactin contains two highly conserved tyrosine residues and a PDZ binding motif. We previously found that phosphorylation of the tyrosine residues is necessary to control the level of Gliotactin at the TCJ. In this study we demonstrate that the phenotypes associated with excess Gliotactin are due to a functional interaction between Gliotactin and Dlg that is dependent on both tyrosine phosphorylation as well as the PDZ binding motif. We further show that elevated levels of Dlg strongly enhance Gliotactin overexpression phenotypes to the point where tissue over-growth is observed. The exhibition of these phenotypes require phosphorylation of Dlg on serine 797, a known Par1 phosphorylation target. Blocking this phosphorylation completely suppresses the cell invasiveness and apoptotic phenotypes associated with Gliotactin overexpression. Additionally, we show that Drosophila JNK acts downstream of Gliotactin and Dlg to mediate the overgrowth and apoptosis caused by the functional interaction of Gliotactin and Dlg.
Asunto(s)
Proteínas de Drosophila/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Membrana Celular/metabolismo , Drosophila melanogaster , Uniones Intercelulares/metabolismoRESUMEN
Peripheral nerve development involves multiple classes of glia that cooperate to form overlapping glial layers paired with the deposition of a surrounding extracellular matrix (ECM). The formation of this tubular structure protects the ensheathed axons from physical and pathogenic damage and from changes in the ionic environment. Integrins, a major family of ECM receptors, play a number of roles in the development of myelinating Schwann cells, one class of glia ensheathing the peripheral nerves of vertebrates. However, the identity and the role of the integrin complexes utilized by the other classes of peripheral nerve glia have not been determined in any animal. Here, we show that, in the peripheral nerves of Drosophila melanogaster, two integrin complexes (αPS2ßPS and αPS3ßPS) are expressed in the different glial layers and form adhesion complexes with integrin-linked kinase and Talin. Knockdown of the common beta subunit (ßPS) using inducible RNAi in all glial cells results in lethality and glial defects. Analysis of integrin complex function in specific glial layers showed that loss of ßPS in the outermost layer (the perineurial glia) results in a failure to wrap the nerve, a phenotype similar to that of Matrix metalloproteinase 2-mediated degradation of the ECM. Knockdown of ßPS integrin in the innermost wrapping glia causes a loss of glial processes around axons. Together, our data suggest that integrins are employed in different glial layers to mediate the development and maintenance of the protective glial sheath in Drosophila peripheral nerves.
Asunto(s)
Integrinas/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Nervios Periféricos/citología , Nervios Periféricos/metabolismo , Animales , Axones/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Matriz Extracelular/metabolismo , Inmunohistoquímica , Integrinas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Talina/genética , Talina/metabolismoRESUMEN
The tricellular junction (TCJ) forms at the convergence of bicellular junctions from three adjacent cells in polarized epithelia and is necessary for maintaining the transepithelial barrier. In the fruitfly Drosophila, the TCJ is generated at the meeting point of bicellular septate junctions. Gliotactin was the first identified component of the TCJ and is necessary for TCJ and septate junction development. Gliotactin is a member of the neuroligin family and associates with the PDZ protein discs large. Beyond this interaction, little is known about the mechanisms underlying Gliotactin localization and function at the TCJ. In this study, we show that Gliotactin is phosphorylated at conserved tyrosine residues, a process necessary for endocytosis and targeting to late endosomes and lysosomes for degradation. Regulation of Gliotactin levels through phosphorylation and endocytosis is necessary as overexpression results in displacement of Gliotactin away from the TCJ throughout the septate junction domain. Excessive Gliotactin in polarized epithelia leads to delamination, paired with subsequent migration, and apoptosis. The apoptosis and the resulting compensatory proliferation resulting from high levels of Gliotactin are mediated by the Drosophila JNK pathway. Therefore, Gliotactin levels within the cell membrane are regulated to ensure correct protein localization and cell survival.
Asunto(s)
Polaridad Celular , Drosophila/metabolismo , Endocitosis , Células Epiteliales/metabolismo , Uniones Intercelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Secuencias de Aminoácidos , Animales , Proliferación Celular , Supervivencia Celular , Drosophila/química , Drosophila/citología , Drosophila/genética , Endosomas/genética , Endosomas/metabolismo , Células Epiteliales/química , Células Epiteliales/citología , Uniones Intercelulares/química , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Fosforilación , Transporte de ProteínasRESUMEN
Septate junctions (SJs), similar to tight junctions, function as transepithelial permeability barriers. Gliotactin (Gli) is a cholinesterase-like molecule that is necessary for blood-nerve barrier integrity, and may, therefore, contribute to SJ development or function. To address this hypothesis, we analyzed Gli expression and the Gli mutant phenotype in Drosophila epithelia. In Gli mutants, localization of SJ markers neurexin-IV, discs large, and coracle are disrupted. Furthermore, SJ barrier function is lost as determined by dye permeability assays. These data suggest that Gli is necessary for SJ formation. Surprisingly, Gli distribution only colocalizes with other SJ markers at tricellular junctions, suggesting that Gli has a unique function in SJ development. Ultrastructural analysis of Gli mutants supports this notion. In contrast to other SJ mutants in which septa are missing, septa are present in Gli mutants, but the junction has an immature morphology. We propose a model, whereby Gli acts at tricellular junctions to bind, anchor, or compact SJ strands apically during SJ development.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/embriología , Epitelio/anomalías , Uniones Intercelulares/patología , Proteínas de la Membrana/deficiencia , Proteínas del Tejido Nervioso/deficiencia , Animales , Animales Modificados Genéticamente , Biomarcadores , Moléculas de Adhesión Celular Neuronal/deficiencia , Moléculas de Adhesión Celular Neuronal/genética , Comunicación Celular/genética , Membrana Celular/metabolismo , Membrana Celular/patología , Membrana Celular/ultraestructura , Permeabilidad de la Membrana Celular/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestructura , Epidermis/anomalías , Epidermis/metabolismo , Epidermis/ultraestructura , Epitelio/metabolismo , Epitelio/ultraestructura , Regulación del Desarrollo de la Expresión Génica/genética , Uniones Intercelulares/metabolismo , Uniones Intercelulares/ultraestructura , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Modelos Animales , Modelos Biológicos , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
The mammalian MAGI proteins play important roles in the maintenance of adherens and tight junctions. The MAGI family of proteins contains modular domains such as WW and PDZ domains necessary for scaffolding of membrane receptors and intracellular signaling components. Loss of MAGI leads to reduced junction stability while overexpression of MAGI can lead to increased adhesion and stabilization of epithelial morphology. However, how Magi regulates junction assembly in epithelia is largely unknown. We investigated the single Drosophila homologue of Magi to study the in vivo role of Magi in epithelial development. Magi is localized at the adherens junction and forms a complex with the polarity proteins, Par3/Bazooka and aPKC. We generated a Magi null mutant and found that Magi null mutants were viable with no detectable morphological defects even though the Magi protein is highly conserved with vertebrate Magi homologues. However, overexpression of Magi resulted in the displacement of Baz/Par3 and aPKC and lead to an increase in the level of PIP3. Interestingly, we found that Magi and Baz functioned in an antagonistic manner to regulate the localization of the apical polarity complex. Maintaining the balance between the level of Magi and Baz is an important determinant of the levels and localization of apical polarity complex.
Asunto(s)
Uniones Adherentes/metabolismo , Polaridad Celular/fisiología , Proteínas de Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Drosophila melanogaster , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Nucleósido-Fosfato Quinasa/genética , Proteína Quinasa C/metabolismo , Uniones Estrechas/metabolismoRESUMEN
A major developmental role of peripheral glia is to mediate sensory axon guidance; however, it is not known whether sensory neurons influence peripheral glial development. To determine whether glia and neurons reciprocally interact during embryonic development, we ablated each cell type by overexpressing the apoptosis gene, grim, and observed the effects on peripheral nervous system (PNS) development. When neurons are ablated, glial defects occur as a secondary effect, and vice versa. Therefore glia and neurons are codependent during embryogenesis. To further explore glial-neuronal interactions, we genetically disrupted glial migration or differentiation and observed the secondary effects on sensory neuron development. Glial migration and ensheathment of PNS axons was blocked by overexpression of activated Rho GTPase, a regulator of actin dynamics. Here, sensory axons extended to the CNS without exhibiting gross pathfinding errors. In contrast, disrupting differentiation by expression of dominant-negative Ras GTPase in glia resulted in major sensory axon pathfinding errors, similar to those seen in glial ablations. Glial overexpression of transgenic components of the epidermal growth factor receptor (EGFR) signaling pathway yielded similar sensory neuron defects and also downregulated the expression of the glial marker Neuroglian. Mutant analysis also suggested that the EGFR ligands Spitz and Vein play roles in peripheral glial development. The observations support a model in which glia express genes necessary for sensory neuron development, and these genes are potentially under the control of the EGFR/Ras signaling pathway.
Asunto(s)
Drosophila/embriología , Factor de Crecimiento Epidérmico , Neuroglía/fisiología , Neuronas Aferentes/fisiología , Sistema Nervioso Periférico/embriología , Animales , Axones/fisiología , Tipificación del Cuerpo , Moléculas de Adhesión Celular Neuronal/metabolismo , Recuento de Células , Muerte Celular , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Proteínas de Drosophila/metabolismo , Embrión no Mamífero , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/metabolismo , Neurregulinas/metabolismo , Neuroglía/citología , Neuronas Aferentes/citología , Sistema Nervioso Periférico/citología , Fenotipo , Transducción de Señal/fisiología , Proteínas ras/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The protein family known as CLAMS (cholinesterase-like adhesion molecules) forms a novel class of heterophilic cell adhesion proteins. Family members are found through a wide range of metazoans and play a role during the development of multiple tissues. The majority of members of this family are transmembrane proteins with an extracellular domain that is conserved with cholinesterases including acetylcholinesterase. Yet all family members lack one or more of the residues that make up the catalytic triad necessary for enzymatic function. Therefore the conserved cholinesterase-like domain is not necessary for enzymatic function but does appear to play a role in heterophilic binding. CLAMS are expressed in a wide array of tissues and most family members appear to play a role in cell adhesion and junction formation. The development of junctions including septate junctions and synaptic junctions require CLAM family members such as Gliotactin and Neuroligins respectively. Modeling of the cholinesterase-like domain reveals that evolutionary changes to the binding pocket of the cholinesterase domain may produce a range of different ligand binding partners for CLAM family members. In this vein, previous chimera experiments and recent work has identified mutations in CLAM family members that affect the structure of the cholinesterase-like domain. These mutant forms affect protein function during the development of specialized junctions and confirm the role of the cholinesterase domain in mediating heterophilic binding.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Colinesterasas/metabolismo , Evolución Molecular , Animales , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular Neuronal , Humanos , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Drosophila gliotactin (Gli) is a 109-kDa transmembrane, cholinesterase-like adhesion molecule (CLAM), expressed in peripheral glia, that is crucial for formation of the blood-nerve barrier. The intracellular portion (Gli-cyt) was cloned and expressed in the cytosolic fraction of Escherichia coli BLR(DE3) at 45 mg/L and purified by Ni-NTA (nitrilotriacetic acid) chromatography. Although migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), under denaturing conditions, was unusually slow, molecular weight determination by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) confirmed that the product was consistent with its theoretical size. Gel filtration chromatography yielded an anomalously large Stokes radius, suggesting a fully unfolded conformation. Circular dichroism (CD) spectroscopy demonstrated that Gli-cyt was >50% unfolded, further suggesting a nonglobular conformation. Finally, 1D-(1)H NMR conclusively demonstrated that Gli-cyt possesses an extended unfolded structure. In addition, Gli-cyt was shown to possess charge and hydrophobic properties characteristic of natively unfolded proteins (i.e., proteins that, when purified, are intrinsically disordered under physiologic conditions in vitro).
Asunto(s)
Proteínas de Drosophila/química , Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Adhesión Celular , Colinesterasas/química , Cromatografía en Gel , Dicroismo Circular , Clonación Molecular , Proteínas de Drosophila/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/aislamiento & purificación , Pliegue de Proteína , Estructura Terciaria de Proteína , Análisis de Secuencia de ProteínaRESUMEN
Glia are integral participants in synaptic physiology, remodeling and maturation from blowflies to humans, yet how glial structure is coordinated with synaptic growth is unknown. To investigate the dynamics of glial development at the Drosophila larval neuromuscular junction (NMJ), we developed a live imaging system to establish the relationship between glia, neuronal boutons, and the muscle subsynaptic reticulum. Using this system we observed processes from two classes of peripheral glia present at the NMJ. Processes from the subperineurial glia formed a blood-nerve barrier around the axon proximal to the first bouton. Processes from the perineurial glial extended beyond the end of the blood-nerve barrier into the NMJ where they contacted synapses and extended across non-synaptic muscle. Growth of the glial processes was coordinated with NMJ growth and synaptic activity. Increasing synaptic size through elevated temperature or the highwire mutation increased the extent of glial processes at the NMJ and conversely blocking synaptic activity and size decreased the presence and size of glial processes. We found that elevated temperature was required during embryogenesis in order to increase glial expansion at the nmj. Therefore, in our live imaging system, glial processes at the NMJ are likely indirectly regulated by synaptic changes to ensure the coordinated growth of all components of the tripartite larval NMJ.
Asunto(s)
Drosophila melanogaster/citología , Neuroglía/citología , Unión Neuromuscular/citología , Animales , Axones/metabolismo , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Larva/citología , Neuronas Motoras/citología , Unión Neuromuscular/embriología , Nervios Periféricos/citología , TemperaturaRESUMEN
Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.
Asunto(s)
Movimiento Celular/fisiología , Drosophila/citología , Ojo/citología , Neuroglía/citología , Animales , Encéfalo/fisiología , Disección , Proteínas Fluorescentes Verdes/biosíntesis , Neuroglía/metabolismoRESUMEN
Glial cells play a wide range of essential roles in both nervous system development and function and has been reviewed recently (Parker and Auld, 2006). Glia provide an insulating sheath, either form or direct the formation of the blood-brain barrier, contribute to ion and metabolite homeostasis and provide guidance cues. Glial function often depends on the ability of glial cells to migrate toward specific locations during nervous system development. Work in nervous system development in insects, in particular in the fruit fly Drosophila melanogaster and the tobacco hornworm Manduca sexta, has provided significant insight into the roles of glia, although the molecular mechanisms underlying glial cell migration are being determined only now. Indeed, many of the processes and mechanisms discovered in these simpler systems have direct parallels in the development of vertebrate nervous systems. In this review, we first examine the developmental contexts in which invertebrate glial cell migration has been observed, we next discuss the characterized molecules required for proper glial cell migration, and we finally discuss future goals to be addressed in the study of glial cell development.
RESUMEN
Glial cells have diverse functions that are necessary for the proper development and function of complex nervous systems. Various insects, primarily the fruit fly Drosophila melanogaster and the moth Manduca sexta, have provided useful models of glial function during development. The present review will outline evidence of glial contributions to embryonic, visual, olfactory and wing development. We will also outline evidence for non-developmental functions of insect glia including blood-brain-barrier formation, homeostatic functions and potential contributions to synaptic function. Where relevant, we will also point out similarities between the functions of insect glia and their vertebrate counterparts.