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1.
PLoS Biol ; 22(3): e3002504, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38478582

RESUMO

Natural ageing is accompanied by a decline in motor, sensory, and cognitive functions, all impacting quality of life. Ageing is also the predominant risk factor for many neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. We need to therefore gain a better understanding of the cellular and physiological processes underlying age-related neuronal decay. However, gaining this understanding is a slow process due to the large amount of time required to age mammalian or vertebrate animal models. Here, we introduce a new cellular model within the Drosophila brain, in which we report classical ageing hallmarks previously observed in the primate brain. These hallmarks include axonal swellings, cytoskeletal decay, a reduction in axonal calibre, and morphological changes arising at synaptic terminals. In the fly brain, these changes begin to occur within a few weeks, ideal to study the underlying mechanisms of ageing. We discovered that the decay of the neuronal microtubule (MT) cytoskeleton precedes the onset of other ageing hallmarks. We showed that the MT-binding factors Tau, EB1, and Shot/MACF1, are necessary for MT maintenance in axons and synapses, and that their functional loss during ageing triggers MT bundle decay, followed by a decline in axons and synaptic terminals. Furthermore, genetic manipulations that improve MT networks slowed down the onset of neuronal ageing hallmarks and confer aged specimens the ability to outperform age-matched controls. Our work suggests that MT networks are a key lesion site in ageing neurons and therefore the MT cytoskeleton offers a promising target to improve neuronal decay in advanced age.


Assuntos
Proteínas de Drosophila , Qualidade de Vida , Animais , Citoesqueleto , Neurônios/patologia , Drosophila , Microtúbulos , Envelhecimento , Mamíferos , Proteínas Associadas aos Microtúbulos , Proteínas de Drosophila/genética
2.
Biochem J ; 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38193346

RESUMO

Cysteine string protein α (CSPα), also known as DNAJC5, is a member of the DnaJ/Hsp40 family of co-chaperones. The name derives from a cysteine-rich domain, palmitoylation of which enables localization to intracellular membranes, notably neuronal synaptic vesicles. Mutations in the DNAJC5 gene that encodes CSPα cause autosomal dominant, adult-onset neuronal ceroid lipofuscinosis (ANCL), a rare neurodegenerative disease. As null mutations in CSP-encoding genes in flies, worms and mice similarly result in neurodegeneration, CSP is evidently an evolutionarily conserved neuroprotective protein. However, the client proteins that CSP chaperones to prevent neurodegeneration remain unclear. Traditional methods for identifying protein-protein interactions such as yeast 2-hybrid and affinity purification approaches are poorly suited to CSP, due to its requirement for membrane anchoring and its tendency to aggregate after cell lysis. Therefore, we employed proximity labelling, which enables identification of interacting proteins in situ in living cells via biotinylation. Neuroendocrine PC12 cell lines stably expressing wild type or L115R ANCL mutant CSP constructs fused to miniTurbo were generated; then the biotinylated proteomes were analysed by liquid chromatographymass spectrometry (LCMS) and validated by western blotting. This confirmed several known CSP-interacting proteins, such as Hsc70 and SNAP-25, but also revealed novel binding proteins, including STXBP1/Munc18-1. Interestingly, some protein interactions (such as Hsc70) were unaffected by the L115R mutation, whereas others (including SNAP-25 and STXBP1/Munc18-1) were inhibited. These results define the CSP interactome in a neuronal model cell line and reveal interactions that are affected by ANCL mutation and hence may contribute to the neurodegeneration seen in patients.

3.
PLoS Genet ; 17(7): e1009647, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34228717

RESUMO

The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the essential microtubule bundles in axons of Drosophila and Xenopus neurons, we show that the plus-end scaffold Eb1, the polymerase XMAP215/Msps and the lattice-binder Tau co-operate interdependently to promote microtubule polymerisation and bundle organisation during axon development and maintenance. Eb1 and XMAP215/Msps promote each other's localisation at polymerising microtubule plus-ends. Tau outcompetes Eb1-binding along microtubule lattices, thus preventing depletion of Eb1 tip pools. The three factors genetically interact and show shared mutant phenotypes: reductions in axon growth, comet sizes, comet numbers and comet velocities, as well as prominent deterioration of parallel microtubule bundles into disorganised curled conformations. This microtubule curling is caused by Eb1 plus-end depletion which impairs spectraplakin-mediated guidance of extending microtubules into parallel bundles. Our demonstration that Eb1, XMAP215/Msps and Tau co-operate during the regulation of microtubule polymerisation and bundle organisation, offers new conceptual explanations for developmental and degenerative axon pathologies.


Assuntos
Axônios/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Axônios/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Neurônios/metabolismo , Polimerização , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Proteínas tau/metabolismo
4.
Semin Cell Dev Biol ; 69: 40-57, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28579450

RESUMO

Spectraplakins are evolutionarily well conserved cytoskeletal linker molecules that are true members of three protein families: plakins, spectrins and Gas2-like proteins. Spectraplakin genes encode at least 7 characteristic functional domains which are combined in a modular fashion into multiple isoforms, and which are responsible for an enormous breadth of cellular functions. These functions are related to the regulation of actin, microtubules, intermediate filaments, intracellular organelles, cell adhesions and signalling processes during the development and maintenance of a wide variety of tissues. To gain a deeper understanding of this enormous functional diversity, invertebrate genetic model organisms, such as the fruit fly Drosophila, can be used to develop concepts and mechanistic paradigms that can inform the investigation in higher animals or humans. Here we provide a comprehensive overview of our current knowledge of the Drosophila spectraplakin Short stop (Shot). We describe its functional domains and isoforms and compare them with those of the mammalian spectraplakins dystonin and MACF1. We then summarise its roles during the development and maintenance of the nervous system, epithelia, oocytes and muscles, taking care to compare and contrast mechanistic insights across these functions in the fly, but especially also with related functions of dystonin and MACF1 in mostly mammalian contexts. We hope that this review will improve the wider appreciation of how work on Drosophila Shot can be used as an efficient strategy to promote the fundamental concepts and mechanisms that underpin spectraplakin functions, with important implications for biomedical research into human disease.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Orientação de Axônios , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Mamíferos/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Homologia de Sequência de Aminoácidos , Sinapses/metabolismo
5.
J Cell Sci ; 130(15): 2506-2519, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28606990

RESUMO

Directed axonal growth depends on correct coordination of the actin and microtubule cytoskeleton in the growth cone. However, despite the relatively large number of proteins implicated in actin-microtubule crosstalk, the mechanisms whereby actin polymerization is coupled to microtubule stabilization and advancement in the peripheral growth cone remained largely unclear. Here, we identified the formin Dishevelled-associated activator of morphogenesis (DAAM) as a novel factor playing a role in concerted regulation of actin and microtubule remodeling in Drosophilamelanogaster primary neurons. In vitro, DAAM binds to F-actin as well as to microtubules and has the ability to crosslink the two filament systems. Accordingly, DAAM associates with the neuronal cytoskeleton, and a significant fraction of DAAM accumulates at places where the actin filaments overlap with that of microtubules. Loss of DAAM affects growth cone and microtubule morphology, and several aspects of microtubule dynamics; and biochemical and cellular assays revealed a microtubule stabilization activity and binding to the microtubule tip protein EB1. Together, these data suggest that, besides operating as an actin assembly factor, DAAM is involved in linking actin remodeling in filopodia to microtubule stabilization during axonal growth.


Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Drosophila/metabolismo , Cones de Crescimento/metabolismo , Microtúbulos/metabolismo , Pseudópodes/metabolismo , Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Pseudópodes/genética
6.
J Cell Sci ; 126(Pt 11): 2331-41, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23729743

RESUMO

The extension of long slender axons is a key process of neuronal circuit formation, both during brain development and regeneration. For this, growth cones at the tips of axons are guided towards their correct target cells by signals. Growth cone behaviour downstream of these signals is implemented by their actin and microtubule cytoskeleton. In the first part of this Commentary, we discuss the fundamental roles of the cytoskeleton during axon growth. We present the various classes of actin- and microtubule-binding proteins that regulate the cytoskeleton, and highlight the important gaps in our understanding of how these proteins functionally integrate into the complex machinery that implements growth cone behaviour. Deciphering such machinery requires multidisciplinary approaches, including genetics and the use of simple model organisms. In the second part of this Commentary, we discuss how the application of combinatorial genetics in the versatile genetic model organism Drosophila melanogaster has started to contribute to the understanding of actin and microtubule regulation during axon growth. Using the example of dystonin-linked neuron degeneration, we explain how knowledge acquired by studying axonal growth in flies can also deliver new understanding in other aspects of neuron biology, such as axon maintenance in higher animals and humans.


Assuntos
Axônios/metabolismo , Citoesqueleto , Proteínas de Drosophila , Cones de Crescimento/metabolismo , Proteínas do Tecido Nervoso , Doenças Neurodegenerativas , Actinas/genética , Actinas/metabolismo , Animais , Axônios/patologia , Citoesqueleto/genética , Citoesqueleto/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Cones de Crescimento/patologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo
7.
J Neurosci ; 32(29): 10035-44, 2012 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-22815517

RESUMO

Axon guidance is a key process during nervous system development and regeneration. One of the best established paradigms to study the mechanisms underlying this process is the axon decision of whether or not to cross the midline in the Drosophila CNS. An essential regulator of that decision is the well conserved Slit-Robo signaling pathway. Slit guidance cues act through Robo receptors to repel axons from the midline. Despite good progress in our knowledge about these proteins, the intracellular mechanisms associated with Robo function remain poorly defined. In this work, we found that the scaffolding protein Canoe (Cno), the Drosophila orthologue of AF-6/Afadin, is essential for Slit-Robo signaling. Cno is expressed along longitudinal axonal pioneer tracts, and longitudinal Robo/Fasciclin2-positive axons aberrantly cross the midline in cno mutant embryos. cno mutant primary neurons show a significant reduction of Robo localized in growth cone filopodia and Cno forms a complex with Robo in vivo. Moreover, the commissureless (comm) phenotype (i.e., lack of commissures due to constitutive surface presentation of Robo in all neurons) is suppressed in comm, cno double-mutant embryos. Specific genetic interactions between cno, slit, robo, and genes encoding other components of the Robo pathway, such as Neurexin-IV, Syndecan, and Rac GTPases, further confirm that Cno functionally interacts with the Slit-Robo pathway. Our data argue that Cno is a novel regulator of the Slit-Robo signaling pathway, crucial for regulating the subcellular localization of Robo and for transducing its signaling to the actin cytoskeleton during axon guidance at the midline.


Assuntos
Axônios/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais/fisiologia , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Quimiotaxia/fisiologia , Citoesqueleto/metabolismo , Drosophila , Proteínas de Drosophila/genética , Feminino , Cones de Crescimento/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Receptores Imunológicos/genética , Proteínas Roundabout
8.
J Neurosci ; 32(46): 16080-94, 2012 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-23152593

RESUMO

The formation of neuronal circuits is a key process of development, laying foundations for behavior. The cellular mechanisms regulating circuit development are not fully understood. Here, we reveal Psidin as an intracellular regulator of Drosophila olfactory system formation. We show that Psidin is required in several classes of olfactory receptor neurons (ORNs) for survival and subsequently for axon guidance. During axon guidance, Psidin functions as an actin regulator and antagonist of Tropomyosin. Accordingly, Psidin-deficient primary neurons in culture display growth cones with significantly smaller lamellipodia. This lamellipodial phenotype, as well as the mistargeting defects in vivo, is suppressed by parallel removal of Tropomyosin. In contrast, Psidin functions as the noncatalytic subunit of the N-acetyltransferase complex B (NatB) to maintain the number of ORNs. Psidin physically binds the catalytic NatB subunit CG14222 (dNAA20) and functionally interacts with it in vivo. We define the dNAA20 interaction domain within Psidin and identify a conserved serine as a candidate for phosphorylation-mediated regulation of NatB complex formation. A phosphomimetic mutation of this serine showed severely reduced binding to dNAA20 in vitro. In vivo, it fully rescued the targeting defect but not the reduction in neuron numbers. In addition, we show that a different amino acid point mutation shows exactly the opposite effect by rescuing only the cell number but not the axon targeting defect. Together, our data suggest that Psidin plays two independent developmental roles via the acquisition of separate signaling pathways, both of which contribute to the formation of olfactory circuits.


Assuntos
Axônios/fisiologia , Proteínas Sanguíneas/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Neurônios/fisiologia , Condutos Olfatórios/fisiologia , Acetiltransferases/metabolismo , Animais , Western Blotting , Contagem de Células , Células Cultivadas , Genótipo , Cones de Crescimento/fisiologia , Imunoprecipitação , Hibridização In Situ , Rede Nervosa/citologia , Rede Nervosa/crescimento & desenvolvimento , Rede Nervosa/fisiologia , Condutos Olfatórios/citologia , Condutos Olfatórios/crescimento & desenvolvimento , Fenótipo , Fosforilação/fisiologia , Pseudópodes/fisiologia , Interferência de RNA , Saccharomyces cerevisiae/metabolismo , Tropomiosina/farmacologia
9.
J Neurosci ; 32(27): 9143-58, 2012 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-22764224

RESUMO

The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homolog Short stop/Shot similarly cause severe axon shortening and microtubule disorganization. How spectraplakins perform these functions is not known. Here we show that axonal growth-promoting roles of Shot require interaction with EB1 (End binding protein) at polymerizing plus ends of microtubules. We show that binding of Shot to EB1 requires SxIP motifs in Shot's C-terminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organization. In addition, we report that Shot localizes along microtubule shafts and stabilizes them against pharmacologically induced depolymerization. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (tip interacting proteins; plus end regulators) and structural MAPs (microtubule-associated proteins). From our data we deduce a model that relates the different features of the spectraplakin C terminus to the two functions of Shot during axonal growth.


Assuntos
Actinas/fisiologia , Axônios/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Proteínas dos Microfilamentos/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Actinas/genética , Motivos de Aminoácidos/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/deficiência , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Inativação de Genes/métodos , Cones de Crescimento/fisiologia , Masculino , Camundongos , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/deficiência , Mutação , Células NIH 3T3 , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/fisiologia , Cultura Primária de Células
10.
Methods Mol Biol ; 2431: 429-449, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35412291

RESUMO

The use of primary neuronal cultures generated from Drosophila tissue provides a powerful model for studies of transport mechanisms. Cultured fly neurons provide similarly detailed subcellular resolution and applicability of pharmacology or fluorescent dyes as mammalian primary neurons. As an experimental advantage for the mechanistic dissection of transport, fly primary neurons can be combined with the fast and highly efficient combinatorial genetics of Drosophila, and genetic tools for the manipulation of virtually every fly gene are readily available. This strategy can be performed in parallel to in vivo transport studies to address relevance of any findings. Here we will describe the generation of primary neuronal cultures from Drosophila embryos and larvae, the use of external fluorescent dyes and genetic tools to label cargo, and the key strategies for live imaging and subsequent analysis.


Assuntos
Transporte Axonal , Drosophila , Animais , Transporte Axonal/fisiologia , Axônios/metabolismo , Drosophila/genética , Corantes Fluorescentes/metabolismo , Cinesinas , Mamíferos , Neurônios
11.
Dev Neurobiol ; 82(4): 288-307, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35333003

RESUMO

Axons are the long and slender processes of neurons constituting the biological cables that wire the nervous system. The growth and maintenance of axons require loose microtubule bundles that extend through their entire length. Understanding microtubule regulation is therefore an essential aspect of axon biology. Key regulators of neuronal microtubules are the spectraplakins, a well-conserved family of cytoskeletal cross-linkers that underlie neuropathies in mouse and humans. Spectraplakin deficiency in mouse or Drosophila causes severe decay of microtubule bundles and reduced axon growth. The underlying mechanisms are best understood for Drosophila's spectraplakin Short stop (Shot) and believed to involve cytoskeletal cross-linkage: Shot's binding to microtubules and Eb1 via its C-terminus has been thoroughly investigated, whereas its F-actin interaction via N-terminal calponin homology (CH) domains is little understood. Here, we have gained new understanding by showing that the F-actin interaction must be finely balanced: altering the properties of F-actin networks or deleting/exchanging Shot's CH domains induces changes in Shot function-with a Lifeact-containing Shot variant causing remarkable remodeling of neuronal microtubules. In addition to actin-microtubule (MT) cross-linkage, we find strong indications that Shot executes redundant MT bundle-promoting roles that are F-actin-independent. We argue that these likely involve the neuronal Shot-PH isoform, which is characterized by a large, unexplored central plakin repeat region (PRR) similarly existing also in mammalian spectraplakins.


Assuntos
Actinas , Proteínas de Drosophila , Actinas/metabolismo , Animais , Axônios/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo
12.
J Neurosci ; 28(49): 13310-9, 2008 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-19052223

RESUMO

The regulation of growth cone actin dynamics is a critical aspect of axonal growth control. Among the proteins that are directly involved in the regulation of actin dynamics, actin nucleation factors play a pivotal role by promoting the formation of novel actin filaments. However, the essential nucleation factors in developing neurons have so far not been clearly identified. Here, we show expression data, and use true loss-of-function analysis and targeted expression of activated constructs to demonstrate that the Drosophila formin DAAM plays a critical role in axonal morphogenesis. In agreement with this finding, we show that dDAAM is required for filopodia formation at axonal growth cones. Our genetic interaction, immunoprecipitation and protein localization studies argue that dDAAM acts in concert with Rac GTPases, Profilin and Enabled during axonal growth regulation. We also show that mouse Daam1 rescues the CNS defects observed in dDAAM mutant flies to a high degree, and vice versa, that Drosophila DAAM induces the formation of neurite-like protrusions when expressed in mouse P19 cells, strongly suggesting that the function of DAAM in developing neurons has been conserved during evolution.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sistema Nervoso Central/embriologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Cones de Crescimento/metabolismo , Neurogênese/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Sequência Conservada/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Evolução Molecular , Feminino , Cones de Crescimento/ultraestrutura , Masculino , Camundongos , Mutação/genética , Vias Neurais/citologia , Vias Neurais/embriologia , Vias Neurais/metabolismo , Neuritos/metabolismo , Neuritos/ultraestrutura , Profilinas/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo
13.
Elife ; 82019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31718774

RESUMO

Cortical collapse factors affect microtubule (MT) dynamics at the plasma membrane. They play important roles in neurons, as suggested by inhibition of axon growth and regeneration through the ARF activator Efa6 in C. elegans, and by neurodevelopmental disorders linked to the mammalian kinesin Kif21A. How cortical collapse factors influence axon growth is little understood. Here we studied them, focussing on the function of Drosophila Efa6 in experimentally and genetically amenable fly neurons. First, we show that Drosophila Efa6 can inhibit MTs directly without interacting molecules via an N-terminal 18 amino acid motif (MT elimination domain/MTED) that binds tubulin and inhibits microtubule growth in vitro and cells. If N-terminal MTED-containing fragments are in the cytoplasm they abolish entire microtubule networks of mouse fibroblasts and whole axons of fly neurons. Full-length Efa6 is membrane-attached, hence primarily blocks MTs in the periphery of fibroblasts, and explorative MTs that have left axonal bundles in neurons. Accordingly, loss of Efa6 causes an increase of explorative MTs: in growth cones they enhance axon growth, in axon shafts they cause excessive branching, as well as atrophy through perturbations of MT bundles. Efa6 over-expression causes the opposite phenotypes. Taken together, our work conceptually links molecular and sub-cellular functions of cortical collapse factors to axon growth regulation and reveals new roles in axon branching and in the prevention of axonal atrophy. Furthermore, the MTED delivers a promising tool that can be used to inhibit MTs in a compartmentalised fashion when fusing it to specifically localising protein domains.


Assuntos
Axônios/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Polimerização , Motivos de Aminoácidos , Animais , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Drosophila/química , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Cones de Crescimento/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Proteínas de Membrana/química , Camundongos , Células NIH 3T3 , Peptídeos/metabolismo , Domínios Proteicos , Pseudópodes/metabolismo
14.
J Neurosci ; 25(1): 78-87, 2005 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-15634769

RESUMO

The phenomenon of pioneer neurons has been known for almost a century, but so far we have little insights into mechanisms and molecules involved. Here, we study the formation of the Drosophila intersegmental motor nerve (ISN). We show that aCC/RP2 and U motor neurons grow together at the leading front of the ISN. Nevertheless, aCC/RP2 neurons are the pioneers, and U neurons are the followers, because only aCC/RP2 neurons effectively influence growth of the ISN. We also show that this influence depends on the neural cell adhesion molecule homolog FasciclinII. First, ablation of aCC/RP2 has a stronger impact on ISN growth than U ablation. Second, strong growth-influencing capabilities of aCC/RP2 are revealed with a stalling approach we used: when aCC/RP2 motor axons are stalled specifically, the entire ISN (including the U neurons) coarrests, demonstrating that aCC/RP2 neurons influence the behavior of U growth cones. In contrast, stalled U neurons do not have the same influence on other ISN motor neurons. The influence on ISN growth requires FasciclinII: targeted expression of FasciclinII in U neurons increases their influence on the ISN, whereas a FasciclinII loss-of-function background reduces ISN coarrest with stalled aCC/RP2 axons. The qualitative differences of both neuron groups are confirmed through our findings that aCC/RP2 growth cones are wider and more complex than those of U neurons. However, U growth cones adopt aCC/RP2-like wider shapes in a FasciclinII loss-of-function background. Therefore, FasciclinII is to a degree required and sufficient for pioneer-follower interactions, but its mode of action cannot be explained merely through an equally bidirectional adhesive interaction.


Assuntos
Axônios/fisiologia , Moléculas de Adesão Celular Neuronais/fisiologia , Neurônios Motores/fisiologia , Animais , Comunicação Celular/fisiologia , Drosophila/anatomia & histologia , Drosophila/embriologia , Proteínas de Drosophila/fisiologia , Cones de Crescimento/fisiologia , Modelos Animais , Modelos Neurológicos , Neurônios Motores/ultraestrutura , Sistema Nervoso/citologia , Sistema Nervoso/embriologia , Vias Neurais/embriologia
15.
Methods Enzymol ; 569: 373-405, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26778568

RESUMO

The cytoskeleton is a dynamic network of filamentous protein polymers required for virtually all cellular processes. It consists of three major classes, filamentous actin (F-actin), intermediate filaments, and microtubules, all displaying characteristic structural properties, functions, cellular distributions, and sets of interacting regulatory proteins. One unique class of proteins, the spectraplakins, bind, regulate, and integrate the functions of all three classes of cytoskeleton proteins. Spectraplakins are giant, evolutionary conserved multidomain proteins (spanning up to 9000 aa) that are true members of the plakin, spectrin, and Gas2-like protein families. They have OMIM-listed disease links to epidermolysis bullosa and hereditary sensory and autonomic neuropathy. Their role in disease is likely underrepresented since studies in model animal systems have revealed critical roles in polarity, morphogenesis, differentiation and maintenance, migration, signaling, and intracellular trafficking in a variety of tissues. This enormous diversity of spectraplakin function is consistent with the numerous isoforms produced from single genomic loci that combine different sets of functional domains in distinct cellular contexts. To study the broad range of functions and complexity of these proteins, Drosophila is a powerful model. Thus, the fly spectraplakin Short stop (Shot) acts as an actin-microtubule linker and plays important roles in many developmental processes, which provide experimentally amenable and relevant contexts in which to study spectraplakin functions. For these studies, a versatile range of relevant experimental resources that facilitate genetics and transgenic approaches, highly refined genomics tools, and an impressive set of spectraplakin-specific genetic and molecular tools are readily available. Here, we use the example of Shot to illustrate how the various tools and strategies available for Drosophila can be employed to decipher and dissect cellular roles and molecular mechanisms of spectraplakins.


Assuntos
Proteínas de Drosophila/genética , Proteínas dos Microfilamentos/genética , Animais , Linhagem Celular , Drosophila , Proteínas de Drosophila/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Células NIH 3T3 , Cultura Primária de Células
16.
Brain Res Bull ; 126(Pt 3): 226-237, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27530065

RESUMO

Axons are the cable-like protrusions of neurons which wire up the nervous system. Polar bundles of microtubules (MTs) constitute their structural backbones and are highways for life-sustaining transport between proximal cell bodies and distal synapses. Any morphogenetic changes of axons during development, plastic rearrangement, regeneration or degeneration depend on dynamic changes of these MT bundles. A key mechanism for implementing such changes is the coordinated polymerisation and depolymerisation at the plus ends of MTs within these bundles. To gain an understanding of how such regulation can be achieved at the cellular level, we provide here an integrated overview of the extensive knowledge we have about the molecular mechanisms regulating MT de/polymerisation. We first summarise insights gained from work in vitro, then describe the machinery which supplies the essential tubulin building blocks, the protein complexes associating with MT plus ends, and MT shaft-based mechanisms that influence plus end dynamics. We briefly summarise the contribution of MT plus end dynamics to important cellular functions in axons, and conclude by discussing the challenges and potential strategies of integrating the existing molecular knowledge into conceptual understanding at the level of axons.


Assuntos
Axônios/metabolismo , Microtúbulos/metabolismo , Animais , Humanos
17.
Elife ; 52016 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-27501441

RESUMO

The mechanisms regulating synapse numbers during development and ageing are essential for normal brain function and closely linked to brain disorders including dementias. Using Drosophila, we demonstrate roles of the microtubule-associated protein Tau in regulating synapse numbers, thus unravelling an important cellular requirement of normal Tau. In this context, we find that Tau displays a strong functional overlap with microtubule-binding spectraplakins, establishing new links between two different neurodegenerative factors. Tau and the spectraplakin Short Stop act upstream of a three-step regulatory cascade ensuring adequate delivery of synaptic proteins. This cascade involves microtubule stability as the initial trigger, JNK signalling as the central mediator, and kinesin-3 mediated axonal transport as the key effector. This cascade acts during development (synapse formation) and ageing (synapse maintenance) alike. Therefore, our findings suggest novel explanations for intellectual disability in Tau deficient individuals, as well as early synapse loss in dementias including Alzheimer's disease.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Cinesinas/genética , Proteínas dos Microfilamentos/genética , Sinapses/genética , Proteínas tau/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Transporte Axonal , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Movimento Celular , Demência/genética , Demência/metabolismo , Demência/patologia , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Cinesinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Neurogênese/genética , Neurônios/metabolismo , Neurônios/ultraestrutura , Transporte Proteico , Transdução de Sinais , Sinapses/metabolismo , Sinapses/ultraestrutura , Proteínas tau/metabolismo
18.
Dev Cell ; 39(2): 267-278, 2016 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-27780041

RESUMO

The axonal wiring molecule Slit and its Round-About (Robo) receptors are conserved regulators of nerve cord patterning. Robo receptors also contribute to wiring brain circuits. Whether molecular mechanisms regulating these signals are modified to fit more complex brain wiring processes is unclear. We investigated the role of Slit and Robo receptors in wiring Drosophila higher-order brain circuits and identified differences in the cellular and molecular mechanisms of Robo/Slit function. First, we find that signaling by Robo receptors in the brain is regulated by the Receptor Protein Tyrosine Phosphatase RPTP69d. RPTP69d increases membrane availability of Robo3 without affecting its phosphorylation state. Second, we detect no midline localization of Slit during brain development. Instead, Slit is enriched in the mushroom body, a neuronal structure covering large areas of the brain. Thus, a divergent molecular mechanism regulates neuronal circuit wiring in the Drosophila brain, partly in response to signals from the mushroom body.


Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Rede Nervosa/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurópilo/metabolismo , Proteínas Tirosina Fosfatases Semelhantes a Receptores/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais , Animais , Axônios/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Epistasia Genética , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Larva/metabolismo , Complexos Multiproteicos/metabolismo , Corpos Pedunculados/metabolismo , Proteínas do Tecido Nervoso/genética , Fenótipo
19.
Elife ; 3: e01699, 2014 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-24755286

RESUMO

Axonal branching allows a neuron to connect to several targets, increasing neuronal circuit complexity. While axonal branching is well described, the mechanisms that control it remain largely unknown. We find that in the Drosophila CNS branches develop through a process of excessive growth followed by pruning. In vivo high-resolution live imaging of developing brains as well as loss and gain of function experiments show that activation of Epidermal Growth Factor Receptor (EGFR) is necessary for branch dynamics and the final branching pattern. Live imaging also reveals that intrinsic asymmetry in EGFR localization regulates the balance between dynamic and static filopodia. Elimination of signaling asymmetry by either loss or gain of EGFR function results in reduced dynamics leading to excessive branch formation. In summary, we propose that the dynamic process of axon branch development is mediated by differential local distribution of signaling receptors. DOI: http://dx.doi.org/10.7554/eLife.01699.001.


Assuntos
Axônios/fisiologia , Plasticidade Neuronal , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , Drosophila , Proteínas de Drosophila/metabolismo , Receptores ErbB/metabolismo , Imagem Óptica , Receptores de Peptídeos de Invertebrados/metabolismo
20.
PLoS One ; 6(3): e18340, 2011 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-21464901

RESUMO

F-actin networks are important structural determinants of cell shape and morphogenesis. They are regulated through a number of actin-binding proteins. The function of many of these proteins is well understood, but very little is known about how they cooperate and integrate their activities in cellular contexts. Here, we have focussed on the cellular roles of actin regulators in controlling filopodial dynamics. Filopodia are needle-shaped, actin-driven cell protrusions with characteristic features that are well conserved amongst vertebrates and invertebrates. However, existing models of filopodia formation are still incomplete and controversial, pieced together from a wide range of different organisms and cell types. Therefore, we used embryonic Drosophila primary neurons as one consistent cellular model to study filopodia regulation. Our data for loss-of-function of capping proteins, enabled, different Arp2/3 complex components, the formin DAAM and profilin reveal characteristic changes in filopodia number and length, providing a promising starting point to study their functional relationships in the cellular context. Furthermore, the results are consistent with effects reported for the respective vertebrate homologues, demonstrating the conserved nature of our Drosophila model system. Using combinatorial genetics, we demonstrate that different classes of nucleators cooperate in filopodia formation. In the absence of Arp2/3 or DAAM filopodia numbers are reduced, in their combined absence filopodia are eliminated, and in genetic assays they display strong functional interactions with regard to filopodia formation. The two nucleators also genetically interact with enabled, but not with profilin. In contrast, enabled shows strong genetic interaction with profilin, although loss of profilin alone does not affect filopodia numbers. Our genetic data support a model in which Arp2/3 and DAAM cooperate in a common mechanism of filopodia formation that essentially depends on enabled, and is regulated through profilin activity at different steps.


Assuntos
Drosophila melanogaster/citologia , Redes Reguladoras de Genes/genética , Cones de Crescimento/metabolismo , Modelos Biológicos , Pseudópodes/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Forma Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Neurônios/citologia , Neurônios/metabolismo , Fenótipo , Profilinas/genética , Profilinas/metabolismo
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