RESUMEN
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.
Asunto(s)
Proteínas de Drosophila , Calidad de Vida , Animales , Citoesqueleto , Neuronas/patología , Drosophila , Microtúbulos , Envejecimiento , Mamíferos , Proteínas Asociadas a Microtúbulos , Proteínas de Drosophila/genéticaRESUMEN
Ciliary defects cause several ciliopathies, some of which have late onset, suggesting cilia are actively maintained. Still, we have a poor understanding of the mechanisms underlying their maintenance. Here, we show Drosophila melanogaster IFT88 (DmIFT88/nompB) continues to move along fully formed sensory cilia. We further identify Inactive, a TRPV channel subunit involved in Drosophila hearing and negative-gravitaxis behaviour, and a yet uncharacterised Drosophila Guanylyl Cyclase 2d (DmGucy2d/CG34357) as DmIFT88 cargoes. We also show DmIFT88 binding to the cyclase´s intracellular part, which is evolutionarily conserved and mutated in several degenerative retinal diseases, is important for the ciliary localisation of DmGucy2d. Finally, acute knockdown of both DmIFT88 and DmGucy2d in ciliated neurons of adult flies caused defects in the maintenance of cilium function, impairing hearing and negative-gravitaxis behaviour, but did not significantly affect ciliary ultrastructure. We conclude that the sensory ciliary function underlying hearing in the adult fly requires an active maintenance program which involves DmIFT88 and at least two of its signalling transmembrane cargoes, DmGucy2d and Inactive.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Cilios/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , AudiciónRESUMEN
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.
Asunto(s)
Axones/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de la Membrana/metabolismo , Microtúbulos/metabolismo , Polimerizacion , Secuencias de Aminoácidos , Animales , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Drosophila/química , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Conos de Crecimiento/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Proteínas de la Membrana/química , Ratones , Células 3T3 NIH , Péptidos/metabolismo , Dominios Proteicos , Seudópodos/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Cinesinas/genética , Proteínas de Microfilamentos/genética , Sinapsis/genética , Proteínas tau/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Transporte Axonal , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Movimiento Celular , Demencia/genética , Demencia/metabolismo , Demencia/patología , Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Cinesinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Neurogénesis/genética , Neuronas/metabolismo , Neuronas/ultraestructura , Transporte de Proteínas , Transducción de Señal , Sinapsis/metabolismo , Sinapsis/ultraestructura , Proteínas tau/metabolismoRESUMEN
The FGFR pathway triggers a wide range of key biological responses. Among others, the Breathless (Btl, Drosophila FGFR1) receptor cascade promotes cell migration during embryonic tracheal system development. However, how the actin cytoskeleton responds to Btl pathway activation to induce cell migration has remained largely unclear. Our recent results shed light into this issue by unveiling a link between the actin-bundling protein Singed (Sn) and the Btl pathway. We showed that the Btl pathway regulates sn, which leads to the stabilization of the actin bundles required for filopodia formation and actin cytoskeleton rearrangement. This regulation contributes to tracheal migration, tracheal branch fusion and tracheal cell elongation. Parallel actin bundles (PABs) are usually cross-linked by more than one actin-bundling protein. Accordingly, we have also shown that sn synergistically interacts with forked (f), another actin crosslinker. In this Extra View we extend f analysis and hypothesize how both actin-bundling proteins may act together to regulate the PABs during tracheal embryonic development. Although both proteins are required for similar tracheal events, we suggest that Sn is essential for actin bundle initiation and stiffening, while F is required for the lengthening and further stabilization of the PABs.
Asunto(s)
Actinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/fisiología , Animales , Drosophila/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Seudópodos/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismoRESUMEN
A key challenge in normal development and in disease is to elucidate the mechanisms of cell migration. Here we approach this question using the tracheal system of Drosophila as a model. Tracheal cell migration requires the Breathless/FGFR pathway; however, how the pathway induces migration remains poorly understood. We find that the Breathless pathway upregulates singed at the tip of tracheal branches, and that this regulation is functionally relevant. singed encodes Drosophila Fascin, which belongs to a conserved family of actin-bundling proteins involved in cancer progression and metastasis upon misregulation. We show that singed is required for filopodia stiffness and proper morphology of tracheal tip cells, defects that correlate with an abnormal actin organisation. We propose that singed-regulated filopodia and cell fronts are required for timely and guided branch migration and for terminal branching and branch fusion. We find that singed requirements rely on its actin-bundling activity controlled by phosphorylation, and that active Singed can promote tip cell features. Furthermore, we find that singed acts in concert with forked, another actin cross-linker. The absence of both cross-linkers further stresses the relevance of tip cell morphology and filopodia for tracheal development. In summary, our results on the one hand reveal a previously undescribed role for forked in the organisation of transient actin structures such as filopodia, and on the other hand identify singed as a new target of Breathless signal, establishing a link between guidance cues, the actin cytoskeleton and tracheal morphogenesis.
Asunto(s)
Proteínas Portadoras/metabolismo , Citoesqueleto/fisiología , Drosophila/embriología , Proteínas de Microfilamentos/metabolismo , Morfogénesis/fisiología , Sistema Respiratorio/embriología , Transducción de Señal/fisiología , Animales , Cartilla de ADN/genética , Proteínas de Drosophila/metabolismo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Hibridación in Situ , Microscopía Confocal , Proteínas Tirosina Quinasas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Imagen de Lapso de TiempoRESUMEN
Filopodia are long and thin finger-like protrusions essential for cell migration. They are formed by parallel actin bundles tightly packed by cell type and context dependent actin-bundling proteins. Our recent work analyzing the role of Fascin during tracheal development in Drosophila has shown that Singed (the Drosophila Fascin homolog) acts as a molecular link between the Branchless (FGF)/Breathless (FGFR) pathway and the actin cytoskeleton. We have reported that the lack of Singed (Sn) leads to wavy and flaccid filopodia due to the disorganization of the tracheal actin cytoskeleton. Here we describe for the first time filopodia breakage in Drosophila, and show that Fascin plays a role in this event. We propose that actin filaments in sn mutant filopodia buckle under membrane pressure due to lower bending stiffness, eventually undergoing breakage. Both Filopodia buckling and breakage would impair correct cell navigation and migration.