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1.
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.


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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
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
13.
Cells ; 9(12)2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33327573

RESUMO

Sperm elongation and nuclear shaping in Drosophila largely depends on the microtubule cytoskeleton that in early spermatids has centrosomal and non-centrosomal origins. We report here an additional γ-tubulin focus localized on the anterior pole of the nucleus in correspondence of the apical end of the perinuclear microtubules that run within the dense complex. The perinuclear microtubules are nucleated by the pericentriolar material, or centriole adjunct, that surrounds the basal body and are retained to play a major role in nuclear shaping. However, we found that both the perinuclear microtubules and the dense complex are present in spermatids lacking centrioles. Therefore, the basal body or the centriole adjunct seem to be dispensable for the organization and assembly of these structures. These observations shed light on a novel localization of γ-tubulin and open a new scenario on the distribution of the microtubules and the organization of the dense complex during early Drosophila spermiogenesis.


Assuntos
Drosophila melanogaster/metabolismo , Microtúbulos/metabolismo , Espermatogênese , Acetilação , Animais , Centríolos/efeitos dos fármacos , Centríolos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/ultraestrutura , Masculino , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Mutação/genética , Nocodazol/farmacologia , Pupa/efeitos dos fármacos , Pupa/metabolismo , Espermátides/efeitos dos fármacos , Espermátides/metabolismo , Espermatócitos/efeitos dos fármacos , Espermatócitos/metabolismo , Espermatogênese/efeitos dos fármacos , Testículo/metabolismo , Testículo/ultraestrutura , Tubulina (Proteína)/metabolismo
14.
PLoS One ; 15(12): e0236495, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382698

RESUMO

The fruit fly Drosophila melanogaster is an important model organism for neuroscience with a wide array of genetic tools that enable the mapping of individual neurons and neural subtypes. Brain templates are essential for comparative biological studies because they enable analyzing many individuals in a common reference space. Several central brain templates exist for Drosophila, but every one is either biased, uses sub-optimal tissue preparation, is imaged at low resolution, or does not account for artifacts. No publicly available Drosophila ventral nerve cord template currently exists. In this work, we created high-resolution templates of the Drosophila brain and ventral nerve cord using the best-available technologies for imaging, artifact correction, stitching, and template construction using groupwise registration. We evaluated our central brain template against the four most competitive, publicly available brain templates and demonstrate that ours enables more accurate registration with fewer local deformations in shorter time.


Assuntos
Encéfalo/anatomia & histologia , Drosophila melanogaster/anatomia & histologia , Tecido Nervoso/anatomia & histologia , Neurônios/ultraestrutura , Animais , Encéfalo/ultraestrutura , Drosophila melanogaster/ultraestrutura , Feminino , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Masculino , Microscopia Confocal , Microscopia Eletrônica , Tecido Nervoso/ultraestrutura
15.
Elife ; 92020 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-33225998

RESUMO

Sustained changes in mood or action require persistent changes in neural activity, but it has been difficult to identify the neural circuit mechanisms that underlie persistent activity and contribute to long-lasting changes in behavior. Here, we show that a subset of Doublesex+ pC1 neurons in the Drosophila female brain, called pC1d/e, can drive minutes-long changes in female behavior in the presence of males. Using automated reconstruction of a volume electron microscopic (EM) image of the female brain, we map all inputs and outputs to both pC1d and pC1e. This reveals strong recurrent connectivity between, in particular, pC1d/e neurons and a specific subset of Fruitless+ neurons called aIPg. We additionally find that pC1d/e activation drives long-lasting persistent neural activity in brain areas and cells overlapping with the pC1d/e neural network, including both Doublesex+ and Fruitless+ neurons. Our work thus links minutes-long persistent changes in behavior with persistent neural activity and recurrent circuit architecture in the female brain.


Assuntos
Encéfalo/fisiologia , Drosophila melanogaster/fisiologia , Vias Neurais/fisiologia , Neurônios/fisiologia , Animais , Encéfalo/ultraestrutura , Corte , Drosophila melanogaster/ultraestrutura , Feminino , Masculino , Microscopia Eletrônica , Atividade Motora/fisiologia , Vias Neurais/ultraestrutura
16.
Development ; 147(20)2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994170

RESUMO

Programmed cell death and consecutive removal of cellular remnants is essential for development. During late stages of Drosophila melanogaster oogenesis, the small somatic follicle cells that surround the large nurse cells promote non-apoptotic nurse cell death, subsequently engulf them, and contribute to the timely removal of nurse cell corpses. Here, we identify a role for Vps13 in the timely removal of nurse cell corpses downstream of developmental programmed cell death. Vps13 is an evolutionarily conserved peripheral membrane protein associated with membrane contact sites and lipid transfer. It is expressed in late nurse cells, and persistent nurse cell remnants are observed when Vps13 is depleted from nurse cells but not from follicle cells. Microscopic analysis revealed enrichment of Vps13 in close proximity to the plasma membrane and the endoplasmic reticulum in nurse cells undergoing degradation. Ultrastructural analysis uncovered the presence of an underlying Vps13-dependent membranous structure in close association with the plasma membrane. The newly identified structure and function suggests the presence of a Vps13-dependent process required for complete degradation of bulky remnants of dying cells.


Assuntos
Apoptose , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Núcleo Celular/metabolismo , Regulação para Baixo , Drosophila melanogaster/ultraestrutura , Retículo Endoplasmático/metabolismo , Feminino , Fertilidade , Mutação/genética , Oogênese , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Folículo Ovariano/ultraestrutura , Fenótipo
17.
Sci Rep ; 10(1): 14574, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32884008

RESUMO

Energy dispersive X-ray spectroscopy (EDS) carried out alongside scanning electron microscopy (SEM) is a common technique for elemental analysis. To investigate "wet" biological specimens, complex pre-treatments are required to stabilize them under the high vacuum conditions of high-resolution SEM. These often produce unwanted artifacts. We have previously reported that the polymerization of natural surface substances on organisms by the electron beam of the SEM setup or by plasma irradiation causes a nano-scale layer to form-called a "NanoSuit"-that can act as a barrier and keep organisms alive and hydrated in a field-emission SEM system. In the study reported herein, we examined the suitability of the NanoSuit method for elemental analyses of biological specimens by EDS. We compared experimental results for living Drosophila larvae and Aloe arborescens specimens prepared by the NanoSuit method and by conventional fixation. The NanoSuit method allowed accurate detection of the elemental compositions at high resolution. By contrast, specimens prepared by the conventional fixation method displayed additional EDS signals corresponding to the elements in the chemicals involved in the fixation process. Our results demonstrate that the NanoSuit method is useful for studying hydrous samples via EDS and SEM, particularly in biological sciences.


Assuntos
Drosophila melanogaster/metabolismo , Elementos Químicos , Larva/metabolismo , Microscopia Eletrônica de Varredura/métodos , Nanotecnologia/métodos , Polímeros/química , Espectrometria por Raios X/métodos , Animais , Drosophila melanogaster/ultraestrutura , Larva/ultraestrutura , Vácuo
18.
Mol Biol Cell ; 31(21): 2331-2347, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32755438

RESUMO

Mitochondria are maternally inherited in many organisms. Mitochondrial morphology and activity regulation is essential for cell survival, differentiation, and migration. An analysis of mitochondrial dynamics and function in morphogenetic events in early metazoan embryogenesis has not been carried out. In our study we find a crucial role of mitochondrial morphology regulation in cell formation in Drosophila embryogenesis. We find that mitochondria are small and fragmented and translocate apically on microtubules and distribute progressively along the cell length during cellularization. Embryos mutant for the mitochondrial fission protein, Drp1 (dynamin-related protein 1), die in embryogenesis and show an accumulation of clustered mitochondria on the basal side in cellularization. Additionally, Drp1 mutant embryos contain lower levels of reactive oxygen species (ROS). ROS depletion was previously shown to decrease myosin II activity. Drp1 loss also leads to myosin II depletion at the membrane furrow, thereby resulting in decreased cell height and larger contractile ring area in cellularization similar to that in myosin II mutants. The mitochondrial morphology and cellularization defects in Drp1 mutants are suppressed by reducing mitochondrial fusion and increasing cytoplasmic ROS in superoxide dismutase mutants. Our data show a key role for mitochondrial morphology and activity in supporting the morphogenetic events that drive cellularization in Drosophila embryos.


Assuntos
Proteínas do Citoesqueleto/fisiologia , Drosophila melanogaster/ultraestrutura , Proteínas de Ligação ao GTP/fisiologia , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial , Morfogênese , Animais , Proteínas do Citoesqueleto/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Mitocôndrias/fisiologia , Miosina Tipo II/metabolismo , Espécies Reativas de Oxigênio/metabolismo
19.
Cell Rep ; 32(5): 107989, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32755582

RESUMO

Amyotrophic lateral sclerosis (ALS) manifests pathological changes in motor neurons and various other cell types. Compared to motor neurons, the contribution of the other cell types to the ALS phenotypes is understudied. G4C2 repeat expansion in C9ORF72 is the most common genetic cause of ALS along with frontotemporal dementia (C9-ALS/FTD), with increasing evidence supporting repeat-encoded poly(GR) in disease pathogenesis. Here, we show in Drosophila muscle that poly(GR) enters mitochondria and interacts with components of the Mitochondrial Contact Site and Cristae Organizing System (MICOS), altering MICOS dynamics and intra-subunit interactions. This impairs mitochondrial inner membrane structure, ion homeostasis, mitochondrial metabolism, and muscle integrity. Similar mitochondrial defects are observed in patient fibroblasts. Genetic manipulation of MICOS components or pharmacological restoration of ion homeostasis with nigericin effectively rescue the mitochondrial pathology and disease phenotypes in both systems. These results implicate MICOS-regulated ion homeostasis in C9-ALS pathogenesis and suggest potential new therapeutic strategies.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Expansão das Repetições de DNA , Demência Frontotemporal/genética , Homeostase , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/patologia , Células HEK293 , Células HeLa , Humanos , Íons , Masculino , Mitocôndrias Musculares/ultraestrutura , Nigericina/farmacologia , Ligação Proteica
20.
Life Sci Alliance ; 3(8)2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32718994

RESUMO

Striated muscle thick filaments are composed of myosin II and several non-myosin proteins. Myosin II's long α-helical coiled-coil tail forms the dense protein backbone of filaments, whereas its N-terminal globular head containing the catalytic and actin-binding activities extends outward from the backbone. Here, we report the structure of thick filaments of the flight muscle of the fruit fly Drosophila melanogaster at 7 Å resolution. Its myosin tails are arranged in curved molecular crystalline layers identical to flight muscles of the giant water bug Lethocerus indicus Four non-myosin densities are observed, three of which correspond to ones found in Lethocerus; one new density, possibly stretchin-mlck, is found on the backbone outer surface. Surprisingly, the myosin heads are disordered rather than ordered along the filament backbone. Our results show striking myosin tail similarity within flight muscle filaments of two insect orders separated by several hundred million years of evolution.


Assuntos
Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Microscopia Crioeletrônica/métodos , Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Relaxamento Muscular/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Sistema Musculoesquelético/metabolismo , Miosina Tipo II/análise , Miosina Tipo II/metabolismo , Miosina Tipo II/ultraestrutura , Miosinas/análise , Miosinas/ultraestrutura , Sarcômeros/metabolismo
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