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1.
Cell ; 187(10): 2574-2594.e23, 2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38729112

RESUMO

High-resolution electron microscopy of nervous systems has enabled the reconstruction of synaptic connectomes. However, we do not know the synaptic sign for each connection (i.e., whether a connection is excitatory or inhibitory), which is implied by the released transmitter. We demonstrate that artificial neural networks can predict transmitter types for presynapses from electron micrographs: a network trained to predict six transmitters (acetylcholine, glutamate, GABA, serotonin, dopamine, octopamine) achieves an accuracy of 87% for individual synapses, 94% for neurons, and 91% for known cell types across a D. melanogaster whole brain. We visualize the ultrastructural features used for prediction, discovering subtle but significant differences between transmitter phenotypes. We also analyze transmitter distributions across the brain and find that neurons that develop together largely express only one fast-acting transmitter (acetylcholine, glutamate, or GABA). We hope that our publicly available predictions act as an accelerant for neuroscientific hypothesis generation for the fly.


Assuntos
Drosophila melanogaster , Microscopia Eletrônica , Neurotransmissores , Sinapses , Animais , Encéfalo/ultraestrutura , Encéfalo/metabolismo , Conectoma , Drosophila melanogaster/ultraestrutura , Drosophila melanogaster/metabolismo , Ácido gama-Aminobutírico/metabolismo , Microscopia Eletrônica/métodos , Redes Neurais de Computação , Neurônios/metabolismo , Neurônios/ultraestrutura , Neurotransmissores/metabolismo , Sinapses/ultraestrutura , Sinapses/metabolismo
2.
Science ; 379(6636): eadd9330, 2023 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-36893230

RESUMO

Brains contain networks of interconnected neurons and so knowing the network architecture is essential for understanding brain function. We therefore mapped the synaptic-resolution connectome of an entire insect brain (Drosophila larva) with rich behavior, including learning, value computation, and action selection, comprising 3016 neurons and 548,000 synapses. We characterized neuron types, hubs, feedforward and feedback pathways, as well as cross-hemisphere and brain-nerve cord interactions. We found pervasive multisensory and interhemispheric integration, highly recurrent architecture, abundant feedback from descending neurons, and multiple novel circuit motifs. The brain's most recurrent circuits comprised the input and output neurons of the learning center. Some structural features, including multilayer shortcuts and nested recurrent loops, resembled state-of-the-art deep learning architectures. The identified brain architecture provides a basis for future experimental and theoretical studies of neural circuits.


Assuntos
Encéfalo , Conectoma , Drosophila melanogaster , Rede Nervosa , Animais , Encéfalo/ultraestrutura , Neurônios/ultraestrutura , Sinapses/ultraestrutura , Drosophila melanogaster/ultraestrutura , Rede Nervosa/ultraestrutura
3.
J Cell Biol ; 221(2)2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-34878519

RESUMO

The neuronal axon is packed with cytoskeletal filaments, membranes, and organelles, many of which move between the cell body and axon tip. Here, we used cryo-electron tomography to survey the internal components of mammalian sensory axons. We determined the polarity of the axonal microtubules (MTs) by combining subtomogram classification and visual inspection, finding MT plus and minus ends are structurally similar. Subtomogram averaging of globular densities in the MT lumen suggests they have a defined structure, which is surprising given they likely contain the disordered protein MAP6. We found the endoplasmic reticulum in axons is tethered to MTs through multiple short linkers. We surveyed membrane-bound cargos and describe unexpected internal features such as granules and broken membranes. In addition, we detected proteinaceous compartments, including numerous virus-like capsid particles. Our observations outline novel features of axonal cargos and MTs, providing a platform for identification of their constituents.


Assuntos
Axônios/ultraestrutura , Compartimento Celular , Microscopia Crioeletrônica , Espaço Intracelular/metabolismo , Mamíferos/metabolismo , Microtúbulos/ultraestrutura , Tomografia , Animais , Axônios/metabolismo , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Gânglios Espinais/metabolismo , Microtúbulos/metabolismo , Análise Multivariada , Proteínas do Tecido Nervoso/metabolismo
4.
Nature ; 599(7883): 147-151, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34616045

RESUMO

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels1. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle2) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions.


Assuntos
Conjuntos de Dados como Assunto , Disseminação de Informação , Microscopia Eletrônica de Varredura , Organelas/ultraestrutura , Animais , Linhagem Celular , Células Cultivadas , Drosophila melanogaster/citologia , Drosophila melanogaster/ultraestrutura , Feminino , Complexo de Golgi/ultraestrutura , Humanos , Interfase , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Varredura/normas , Microtúbulos/ultraestrutura , Neuroglia/ultraestrutura , Neurônios/ultraestrutura , Publicação de Acesso Aberto , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/ultraestrutura , Ribossomos/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/ultraestrutura
5.
Cell Rep ; 36(9): 109541, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34469730

RESUMO

The regulation of lipid homeostasis is not well understood. Using forward genetic screening, we demonstrate that the loss of dTBC1D22, an essential gene that encodes a Tre2-Bub2-Cdc16 (TBC) domain-containing protein, results in lipid droplet accumulation in multiple tissues. We observe that dTBC1D22 interacts with Rab40 and exhibits GTPase activating protein (GAP) activity. Overexpression of either the GTP- or GDP-binding-mimic form of Rab40 results in lipid droplet accumulation. We observe that Rab40 mutant flies are defective in lipid mobilization. The lipid depletion induced by overexpression of Brummer, a triglyceride lipase, is dependent on Rab40. Rab40 mutant flies exhibit decreased lipophagy and small size of autolysosomal structures, which may be due to the defective Golgi functions. Finally, we demonstrate that Rab40 physically interacts with Lamp1, and Rab40 is required for the distribution of Lamp1 during starvation. We propose that dTBC1D22 functions as a GAP for Rab40 to regulate lipophagy.


Assuntos
Autofagia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Olho/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Metabolismo dos Lipídeos , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Olho/ultraestrutura , Proteínas Ativadoras de GTPase/genética , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células HeLa , Homeostase , Humanos , Lipase/genética , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Mutação , Proteínas rab de Ligação ao GTP/genética
6.
Elife ; 102021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34523418

RESUMO

Insects have evolved diverse and remarkable strategies for navigating in various ecologies all over the world. Regardless of species, insects share the presence of a group of morphologically conserved neuropils known collectively as the central complex (CX). The CX is a navigational center, involved in sensory integration and coordinated motor activity. Despite the fact that our understanding of navigational behavior comes predominantly from ants and bees, most of what we know about the underlying neural circuitry of such behavior comes from work in fruit flies. Here, we aim to close this gap, by providing the first comprehensive map of all major columnar neurons and their projection patterns in the CX of a bee. We find numerous components of the circuit that appear to be highly conserved between the fly and the bee, but also highlight several key differences which are likely to have important functional ramifications.


Bumblebees forage widely for pollen and nectar from flowers, sometimes travelling kilometers away from their nest, but they can somehow always find their way home in a nearly straight line. These insects have been known to return to their nest from new locations almost 10 kilometers away. This homing ability is a complex neurological feat and requires the brain to combine several processes, including observing the external world, controlling bodily movements and drawing on memory. While the navigational behavior of bees has been well-studied, the neuronal circuitry behind it has not. Unfortunately, most of what is known about insects' brain activity comes from studies in species such as locusts or fruit flies. In these species, a region of the brain known as the central complex has been shown to have an essential role in homing behaviors. However, it is unknown how similar the central complex of bumblebees might be to fruit flies' or locusts', or how these differences may affect navigational abilities. Sayre et al. obtained images of thin slices of the bumblebee central complex using a technique called block-face electron microscopy, which produces high-resolution image volumes. These images were used to obtain a three-dimensional map of over 1300 neurons. This cellular atlas showed that key aspects of the central complex are nearly identical between flies and bumblebees, including the internal compass that monitors what direction the insect is travelling in. However, hundreds of millions of years of independent evolution have resulted in some differences. These were found in neurons possibly involved in forming memories of the directions and lengths of travelled paths, and in the circuits that use such vector memories to steer the insects towards their targets. Sayre et al. propose that these changes underlie bees' impressive ability to navigate. These results help explain how the structure of insects' brains can determine homing abilities. The insights gained could be used to develop efficient autonomous navigation systems, which are challenging to build and require a lot more processing power than offered by a small part of an insect brain.


Assuntos
Abelhas/fisiologia , Comportamento Animal , Conectoma , Voo Animal , Vias Neurais/fisiologia , Neurópilo/fisiologia , Comportamento Espacial , Animais , Abelhas/ultraestrutura , Drosophila melanogaster/fisiologia , Drosophila melanogaster/ultraestrutura , Vias Neurais/ultraestrutura , Neurópilo/ultraestrutura , Especificidade da Espécie
7.
Elife ; 102021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34448452

RESUMO

Skeletal muscles are composed of hundreds of multinucleated muscle fibers (myofibers) whose myonuclei are regularly positioned all along the myofiber's periphery except the few ones clustered underneath the neuromuscular junction (NMJ) at the synaptic zone. This precise myonuclei organization is altered in different types of muscle disease, including centronuclear myopathies (CNMs). However, the molecular machinery regulating myonuclei position and organization in mature myofibers remains largely unknown. Conversely, it is also unclear how peripheral myonuclei positioning is lost in the related muscle diseases. Here, we describe the microtubule-associated protein, MACF1, as an essential and evolutionary conserved regulator of myonuclei positioning and maintenance, in cultured mammalian myotubes, in Drosophila muscle, and in adult mammalian muscle using a conditional muscle-specific knockout mouse model. In vitro, we show that MACF1 controls microtubules dynamics and contributes to microtubule stabilization during myofiber's maturation. In addition, we demonstrate that MACF1 regulates the microtubules density specifically around myonuclei, and, as a consequence, governs myonuclei motion. Our in vivo studies show that MACF1 deficiency is associated with alteration of extra-synaptic myonuclei positioning and microtubules network organization, both preceding NMJ fragmentation. Accordingly, MACF1 deficiency results in reduced muscle excitability and disorganized triads, leaving voltage-activated sarcoplasmic reticulum Ca2+ release and maximal muscle force unchanged. Finally, adult MACF1-KO mice present an improved resistance to fatigue correlated with a strong increase in mitochondria biogenesis.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Junção Neuromuscular/metabolismo , Biogênese de Organelas , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Acoplamento Excitação-Contração , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Microtúbulos/genética , Microtúbulos/ultraestrutura , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/ultraestrutura , Fadiga Muscular , Fibras Musculares Esqueléticas/ultraestrutura , Força Muscular , Mioblastos Esqueléticos/ultraestrutura , Junção Neuromuscular/genética , Junção Neuromuscular/ultraestrutura , Fatores de Tempo
8.
Cells ; 10(8)2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34440763

RESUMO

Among the morphological processes that characterize the early stages of Drosophila oogenesis, the dynamic of the centrioles deserves particular attention. We re-examined the architecture and the distribution of the centrioles within the germarium and early stages of the vitellarium. We found that most of the germ cell centrioles diverge from the canonical model and display notable variations in size. Moreover, duplication events were frequently observed within the germarium in the absence of DNA replication. Finally, we report the presence of an unusually long centriole that is first detected in the cystoblast and is always associated with the developing oocyte. This centriole is directly inherited after the asymmetric division of the germline stem cells and persists during the process of oocyte selection, thus already representing a marker for oocyte identification at the beginning of its formation and during the ensuing developmental stages.


Assuntos
Centríolos/fisiologia , Drosophila melanogaster/fisiologia , Oócitos/fisiologia , Oogênese , Animais , Centríolos/genética , Centríolos/ultraestrutura , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Feminino , Microscopia Eletrônica de Transmissão , Oócitos/ultraestrutura , Fatores de Tempo
9.
Dev Cell ; 56(12): 1700-1711.e8, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34081909

RESUMO

What regulates the spatiotemporal distribution of cell elimination in tissues remains largely unknown. This is particularly relevant for epithelia with high rates of cell elimination where simultaneous death of neighboring cells could impair epithelial sealing. Here, using the Drosophila pupal notum (a single-layer epithelium) and a new optogenetic tool to trigger caspase activation and cell extrusion, we first showed that death of clusters of at least three cells impaired epithelial sealing; yet, such clusters were almost never observed in vivo. Accordingly, statistical analysis and simulations of cell death distribution highlighted a transient and local protective phase occurring near every cell death. This protection is driven by a transient activation of ERK in cells neighboring extruding cells, which inhibits caspase activation and prevents elimination of cells in clusters. This suggests that the robustness of epithelia with high rates of cell elimination is an emerging property of local ERK feedback.


Assuntos
Caspases/genética , Drosophila melanogaster/genética , Células Epiteliais/ultraestrutura , Epitélio/crescimento & desenvolvimento , Animais , Apoptose/genética , Morte Celular/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/ultraestrutura , Células Epiteliais/citologia , Epitélio/ultraestrutura , Sistema de Sinalização das MAP Quinases/genética , Pupa/genética , Pupa/crescimento & desenvolvimento , Pupa/ultraestrutura , Análise de Célula Única
10.
Elife ; 102021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34085637

RESUMO

Neuroendocrine systems in animals maintain organismal homeostasis and regulate stress response. Although a great deal of work has been done on the neuropeptides and hormones that are released and act on target organs in the periphery, the synaptic inputs onto these neuroendocrine outputs in the brain are less well understood. Here, we use the transmission electron microscopy reconstruction of a whole central nervous system in the Drosophila larva to elucidate the sensory pathways and the interneurons that provide synaptic input to the neurosecretory cells projecting to the endocrine organs. Predicted by network modeling, we also identify a new carbon dioxide-responsive network that acts on a specific set of neurosecretory cells and that includes those expressing corazonin (Crz) and diuretic hormone 44 (Dh44) neuropeptides. Our analysis reveals a neuronal network architecture for combinatorial action based on sensory and interneuronal pathways that converge onto distinct combinations of neuroendocrine outputs.


Assuntos
Conectoma , Drosophila melanogaster/ultraestrutura , Interneurônios/ultraestrutura , Sistemas Neurossecretores/ultraestrutura , Células Receptoras Sensoriais/ultraestrutura , Sinapses/ultraestrutura , Animais , Animais Geneticamente Modificados , Dióxido de Carbono/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Hormônios de Inseto/genética , Hormônios de Inseto/metabolismo , Interneurônios/metabolismo , Microscopia Eletrônica de Transmissão , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Sistemas Neurossecretores/metabolismo , Células Receptoras Sensoriais/metabolismo , Sinapses/metabolismo
11.
J Cell Biol ; 220(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34160561

RESUMO

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)-SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.


Assuntos
Drosophila melanogaster/ultraestrutura , Células da Granulosa/ultraestrutura , Glândulas Mamárias Animais/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Coloração e Rotulagem/métodos , Células Tecais/ultraestrutura , Traqueia/ultraestrutura , Animais , Drosophila melanogaster/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Feminino , Expressão Gênica , Genes Reporter , Células da Granulosa/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Larva/metabolismo , Larva/ultraestrutura , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Glândulas Mamárias Animais/metabolismo , Camundongos , Microscopia Eletrônica de Varredura/instrumentação , Organoides/metabolismo , Organoides/ultraestrutura , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Células Tecais/metabolismo , Traqueia/metabolismo , Fluxo de Trabalho , Proteína Vermelha Fluorescente
12.
J Biol Chem ; 297(1): 100804, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34044018

RESUMO

The functional amyloid Orb2 belongs to the cytoplasmic polyadenylation element binding (CPEB) protein family and plays an important role in long-term memory formation in Drosophila. The Orb2 domain structure combines RNA recognition motifs with low-complexity sequences similar to many RNA-binding proteins shown to form protein droplets via liquid-liquid phase separation (LLPS) in vivo and in vitro. This similarity suggests that Orb2 might also undergo LLPS. However, cellular Orb2 puncta have very little internal protein mobility, and Orb2 forms fibrils in Drosophila brains that are functionally active indicating that LLPS might not play a role for Orb2. In the present work, we reconcile these two views on Orb2 droplet formation. Using fluorescence microscopy, we show that soluble Orb2 can indeed phase separate into protein droplets. However, fluorescence recovery after photobleaching (FRAP) data shows that these droplets have either no or only an extremely short-lived liquid phase and appear maturated right after formation. Orb2 fragments that lack the C-terminal RNA-binding domain (RBD) form fibrils out of these droplets. Solid-state NMR shows that these fibrils have well-ordered static domains in addition to the Gln/His-rich fibril core. Further, we find that full-length Orb2B, which is by far the major component of Orb2 fibrils in vivo, does not transition into fibrils but remains in the droplet phase. Together, our data suggest that phase separation might play a role in initiating the formation of functional Orb2 fibrils.


Assuntos
Amiloide/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Sequência de Aminoácidos , Amiloide/ultraestrutura , Animais , Benzotiazóis/metabolismo , Isótopos de Carbono , Proteínas de Drosophila/química , Drosophila melanogaster/ultraestrutura , Fluorescência , Concentração Osmolar , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Poliadenilação e Clivagem de mRNA/química
13.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34019080

RESUMO

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer's disease, and suggest that misregulated EV traffic may be an underlying defect.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Vesículas Extracelulares/metabolismo , Terminações Pré-Sinápticas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Animais , Animais Geneticamente Modificados , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Vesículas Extracelulares/genética , Vesículas Extracelulares/ultraestrutura , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Terminações Pré-Sinápticas/ultraestrutura , Transporte Proteico , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética
14.
Elife ; 102021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33847563

RESUMO

Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here, we show by cell-free reconstitution that binding to flat lipid membranes requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septin hexamers form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12- to 18-nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4-nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.


Assuntos
Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Lipídeos de Membrana/metabolismo , Septinas/metabolismo , Animais , Animais Geneticamente Modificados , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Proteínas de Drosophila/genética , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Humanos , Bicamadas Lipídicas , Lipídeos de Membrana/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/ultraestrutura , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Técnicas de Microbalança de Cristal de Quartzo , Septinas/genética , Septinas/ultraestrutura , Relação Estrutura-Atividade
15.
Cell Rep ; 34(13): 108918, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33789114

RESUMO

Membrane curvature recruits Bin-Amphiphysin-Rvs (BAR)-domain proteins and induces local F-actin assembly, which further modifies the membrane curvature and dynamics. The downstream molecular pathway in vivo is still unclear. Here, we show that a tubular endomembrane scaffold supported by contractile actomyosin stabilizes the somatic cyst cell membrane folded around rigid spermatid heads during the final stages of sperm maturation in Drosophila testis. The structure resembles an actin "basket" covering the bundle of spermatid heads. Genetic analyses suggest that the actomyosin organization is nucleated exclusively by the formins - Diaphanous and Dishevelled Associated Activator of Morphogenesis (DAAM) - downstream of Rho1, which is recruited by the BAR-domain protein Amphiphysin. Actomyosin activity at the actin basket gathers the spermatid heads into a compact bundle and resists the somatic cell invasion by intruding spermatids. These observations reveal a distinct response mechanism of actin-membrane interactions, which generates a cell-adhesion-like strategy through active clamping.


Assuntos
Actomiosina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Espermátides/metabolismo , Actinas/química , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Membrana Celular/metabolismo , Drosophila melanogaster/ultraestrutura , Forminas/metabolismo , Masculino , Proteínas rho de Ligação ao GTP/metabolismo
16.
Cell Rep ; 34(12): 108875, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33761355

RESUMO

The maintenance of mitochondrial homeostasis requires PTEN-induced kinase 1 (PINK1)-dependent mitophagy, and mutations in PINK1 are associated with Parkinson's disease (PD). PINK1 is also downregulated in tumor cells with PTEN mutations. However, there is limited information concerning the role of PINK1 in tissue growth and tumorigenesis. Here, we show that the loss of pink1 caused multiple growth defects independent of its pathological target, Parkin. Moreover, knocking down pink1 in muscle cells induced hyperglycemia and limited systemic organismal growth by the induction of Imaginal morphogenesis protein-Late 2 (ImpL2). Similarly, disrupting PTEN activity in multiple tissues impaired systemic growth by reducing pink1 expression, resembling wasting-like syndrome in cancer patients. Furthermore, the re-expression of PINK1 fully rescued defects in carbohydrate metabolism and systemic growth induced by the tissue-specific pten mutations. Our data suggest a function for PINK1 in regulating systemic growth in Drosophila and shed light on its role in wasting in the context of PTEN mutations.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Mutação/genética , PTEN Fosfo-Hidrolase/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Metabolismo dos Carboidratos , Proliferação de Células , Regulação para Baixo/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/ultraestrutura , Genes Supressores de Tumor , Hiperglicemia/patologia , Insulina/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Músculos/patologia , Neuroglia/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo
17.
Cell Rep ; 34(11): 108871, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33730583

RESUMO

The formation and consolidation of memories are complex phenomena involving synaptic plasticity, microcircuit reorganization, and the formation of multiple representations within distinct circuits. To gain insight into the structural aspects of memory consolidation, we focus on the calyx of the Drosophila mushroom body. In this essential center, essential for olfactory learning, second- and third-order neurons connect through large synaptic microglomeruli, which we dissect at the electron microscopy level. Focusing on microglomeruli that respond to a specific odor, we reveal that appetitive long-term memory results in increased numbers of precisely those functional microglomeruli responding to the conditioned odor. Hindering memory consolidation by non-coincident presentation of odor and reward, by blocking protein synthesis, or by including memory mutants suppress these structural changes, revealing their tight correlation with the process of memory consolidation. Thus, olfactory long-term memory is associated with input-specific structural modifications in a high-order center of the fly brain.


Assuntos
Drosophila melanogaster/fisiologia , Consolidação da Memória/fisiologia , Corpos Pedunculados/inervação , Rede Nervosa/fisiologia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/ultraestrutura , Consolidação da Memória/efeitos dos fármacos , Memória de Longo Prazo/efeitos dos fármacos , Corpos Pedunculados/efeitos dos fármacos , Corpos Pedunculados/ultraestrutura , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/ultraestrutura , Plasticidade Neuronal/efeitos dos fármacos , Odorantes , Ácidos Oleicos/farmacologia , Feromônios/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Sinapses/ultraestrutura
18.
Cell ; 184(3): 759-774.e18, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33400916

RESUMO

To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to acquire a synapse-resolution dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we studied neuronal networks that control leg and wing movements by reconstructing all 507 motor neurons that control the limbs. We show that a specific class of leg sensory neurons synapses directly onto motor neurons with the largest-caliber axons on both sides of the body, representing a unique pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM more accessible and affordable to the scientific community.


Assuntos
Envelhecimento/fisiologia , Drosophila melanogaster/ultraestrutura , Microscopia Eletrônica de Transmissão , Neurônios Motores/ultraestrutura , Células Receptoras Sensoriais/ultraestrutura , Animais , Automação , Conectoma , Extremidades/inervação , Nervos Periféricos/ultraestrutura , Sinapses/ultraestrutura
19.
Nat Cell Biol ; 23(2): 136-146, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33495633

RESUMO

Cell competition allows winner cells to eliminate less fit loser cells in tissues. In Minute cell competition, cells with a heterozygous mutation in ribosome genes, such as RpS3+/- cells, are eliminated by wild-type cells. How cells are primed as losers is partially understood and it has been proposed that reduced translation underpins the loser status of ribosome mutant, or Minute, cells. Here, using Drosophila, we show that reduced translation does not cause cell competition. Instead, we identify proteotoxic stress as the underlying cause of the loser status for Minute competition and competition induced by mahjong, an unrelated loser gene. RpS3+/- cells exhibit reduced autophagic and proteasomal flux, accumulate protein aggregates and can be rescued from competition by improving their proteostasis. Conversely, inducing proteotoxic stress is sufficient to turn otherwise wild-type cells into losers. Thus, we propose that tissues may preserve their health through a proteostasis-based mechanism of cell competition and cell selection.


Assuntos
Competição entre as Células , Drosophila melanogaster/citologia , Proteínas/toxicidade , Estresse Fisiológico , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Competição entre as Células/efeitos dos fármacos , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos , Biossíntese de Proteínas/efeitos dos fármacos , Proteostase/efeitos dos fármacos , Proteínas Ribossômicas/metabolismo , Estresse Fisiológico/efeitos dos fármacos
20.
J Cell Biol ; 220(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33263729

RESUMO

Mechanoreceptor cells develop a specialized cytoskeleton that plays structural and sensory roles at the site of mechanotransduction. However, little is known about how the cytoskeleton is organized and formed. Using electron tomography and live-cell imaging, we resolve the 3D structure and dynamics of the microtubule-based cytoskeleton in fly campaniform mechanosensory cilia. Investigating the formation of the cytoskeleton, we find that katanin p60-like 1 (kat-60L1), a neuronal type of microtubule-severing enzyme, serves two functions. First, it amplifies the mass of microtubules to form the dense microtubule arrays inside the sensory cilia. Second, it generates short microtubules that are required to build the nanoscopic cytoskeleton at the mechanotransduction site. Additional analyses further reveal the functional roles of Patronin and other potential factors in the local regulatory network. In all, our results characterize the specialized cytoskeleton in fly external mechanosensory cilia at near-molecular resolution and provide mechanistic insights into how it is formed.


Assuntos
Proteínas de Drosophila/metabolismo , Katanina/metabolismo , Mecanotransdução Celular , Animais , Polaridade Celular , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Extremidades/fisiologia , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Biológicos , Organelas/metabolismo , Organelas/ultraestrutura , Receptores de Superfície Celular/metabolismo
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