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1.
PLoS Genet ; 17(10): e1009871, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34714823

RESUMO

Kohlschütter-Tönz syndrome (KTS) manifests as neurological dysfunctions, including early-onset seizures. Mutations in the citrate transporter SLC13A5 are associated with KTS, yet their underlying mechanisms remain elusive. Here, we report that a Drosophila SLC13A5 homolog, I'm not dead yet (Indy), constitutes a neurometabolic pathway that suppresses seizure. Loss of Indy function in glutamatergic neurons caused "bang-induced" seizure-like behaviors. In fact, glutamate biosynthesis from the citric acid cycle was limiting in Indy mutants for seizure-suppressing glutamate transmission. Oral administration of the rate-limiting α-ketoglutarate in the metabolic pathway rescued low glutamate levels in Indy mutants and ameliorated their seizure-like behaviors. This metabolic control of the seizure susceptibility was mapped to a pair of glutamatergic neurons, reversible by optogenetic controls of their activity, and further relayed onto fan-shaped body neurons via the ionotropic glutamate receptors. Accordingly, our findings reveal a micro-circuit that links neural metabolism to seizure, providing important clues to KTS-associated neurodevelopmental deficits.


Assuntos
Ciclo do Ácido Cítrico/fisiologia , Ácido Glutâmico/metabolismo , Convulsões/metabolismo , Animais , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico/genética , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ácido Glutâmico/genética , Masculino , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Mutação/genética , Neurônios/metabolismo , Convulsões/genética , Simportadores/genética , Simportadores/metabolismo
2.
PLoS Genet ; 17(10): e1009551, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34714826

RESUMO

Rhodopsins are light-detecting proteins coupled with retinal chromophores essential for visual function. Coincidentally, dysfunctional Rhodopsin homeostasis underlies retinal degeneration in humans and model organisms. Drosophila ninaEG69D mutant is one such example, where the encoded Rh1 protein imposes endoplasmic reticulum (ER) stress and causes light-dependent retinal degeneration. The underlying reason for such light-dependency remains unknown. Here, we report that Drosophila fatty acid binding protein (fabp) is a gene induced in ninaEG69D/+ photoreceptors, and regulates light-dependent Rhodopsin-1 (Rh1) protein clearance and photoreceptor survival. Specifically, our photoreceptor-specific gene expression profiling study in ninaEG69D/+ flies revealed increased expression of fabp together with other genes that control light-dependent Rh1 protein degradation. fabp induction in ninaEG69D photoreceptors required vitamin A and its transporter genes. In flies reared under light, loss of fabp caused an accumulation of Rh1 proteins in cytoplasmic vesicles. The increase in Rh1 levels under these conditions was dependent on Arrestin2 that mediates feedback inhibition of light-activated Rh1. fabp mutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-induced Rh1 degradation. These observations reveal a previously unrecognized link between light-dependent Rh1 proteostasis and the ER-stress imposing ninaEG69D mutant that cause retinal degeneration.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Animais , Mutação/genética , Fenótipo , Retina/metabolismo , Degeneração Retiniana/metabolismo , Transcriptoma/genética
3.
Cells ; 10(10)2021 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-34685757

RESUMO

Neuronal dendrites receive, integrate, and process numerous inputs and therefore serve as the neuron's "antennae". Dendrites display extreme morphological diversity across different neuronal classes to match the neuron's specific functional requirements. Understanding how this structural diversity is specified is therefore important for shedding light on information processing in the healthy and diseased nervous system. Popular models for in vivo studies of dendrite differentiation are the four classes of dendritic arborization (c1da-c4da) neurons of Drosophila larvae with their class-specific dendritic morphologies. Using da neurons, a combination of live-cell imaging and computational approaches have delivered information on the distinct phases and the time course of dendrite development from embryonic stages to the fully developed dendritic tree. With these data, we can start approaching the basic logic behind differential dendrite development. A major role in the definition of neuron-type specific morphologies is played by dynamic actin-rich processes and the regulation of their properties. This review presents the differences in the growth programs leading to morphologically different dendritic trees, with a focus on the key role of actin modulatory proteins. In addition, we summarize requirements and technological progress towards the visualization and manipulation of such actin regulators in vivo.


Assuntos
Actinas/metabolismo , Dendritos/metabolismo , Drosophila/metabolismo , Animais , Diferenciação Celular
4.
J Cell Sci ; 134(20)2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34553767

RESUMO

Proper regulation of microtubule (MT) stability and dynamics is vital for essential cellular processes, including axonal transportation and synaptic growth and remodeling in neurons. In the present study, we demonstrate that the Drosophila ankyrin repeat and KH domain-containing protein Mask negatively affects MT stability in both larval muscles and motor neurons. In larval muscles, loss-of-function of mask increases MT polymer length, and in motor neurons, loss of mask function results in overexpansion of the presynaptic terminal at the larval neuromuscular junctions (NMJs). mask genetically interacts with stathmin (stai), a neuronal modulator of MT stability, in the regulation of axon transportation and synaptic terminal stability. Our structure-function analysis of Mask revealed that its ankyrin repeats domain-containing N-terminal portion is sufficient to mediate Mask's impact on MT stability. Furthermore, we discovered that Mask negatively regulates the abundance of the MT-associated protein Jupiter in motor neuron axons, and that neuronal knocking down of Jupiter partially suppresses mask loss-of-function phenotypes at the larval NMJs. Taken together, our studies demonstrate that Mask is a novel regulator for MT stability, and such a role of Mask requires normal function of Jupiter.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Repetição de Anquirina , Proteínas de Ligação a DNA/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Microtúbulos/metabolismo , Neurônios Motores/metabolismo
5.
Elife ; 102021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34499028

RESUMO

The ubiquitous Ca2+ sensor calmodulin (CaM) binds and regulates many proteins, including ion channels, CaM kinases, and calcineurin, according to Ca2+-CaM levels. What regulates neuronal CaM levels, is, however, unclear. CaM-binding transcription activators (CAMTAs) are ancient proteins expressed broadly in nervous systems and whose loss confers pleiotropic behavioral defects in flies, mice, and humans. Using Caenorhabditis elegans and Drosophila, we show that CAMTAs control neuronal CaM levels. The behavioral and neuronal Ca2+ signaling defects in mutants lacking camt-1, the sole C. elegans CAMTA, can be rescued by supplementing neuronal CaM. CAMT-1 binds multiple sites in the CaM promoter and deleting these sites phenocopies camt-1. Our data suggest CAMTAs mediate a conserved and general mechanism that controls neuronal CaM levels, thereby regulating Ca2+ signaling, physiology, and behavior.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Proteínas de Drosophila/metabolismo , Neurônios/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Calcineurina/metabolismo , Cálcio/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Edição de Genes , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Ligação Proteica , Transdução de Sinais , Transativadores/genética , Transcriptoma
6.
G3 (Bethesda) ; 11(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34544118

RESUMO

DNA topoisomerase I (Top1) maintains chromatin conformation during transcription. While Top1 is not essential in simple eukaryotic organisms such as yeast, it is required for the development of multicellular organisms. In fact, tissue and cell-type-specific functions of Top1 have been suggested in the fruit fly Drosophila. A better understanding of Top1's function in the context of development is important as Top1 inhibitors are among the most widely used anticancer drugs. As a step toward such a better understanding, we studied its localization in live cells of Drosophila. Consistent with prior results, Top1 is highly enriched at the nucleolus in transcriptionally active polyploid cells, and this enrichment responds to perturbation of transcription. In diploid cells, we uncovered evidence for Top1 foci formation at genomic regions not limited to the active rDNA locus, suggestive of novel regulation of Top1 recruitment. In the male germline, Top1 is highly enriched at the paired rDNA loci on sex chromosomes suggesting that it might participate in regulating their segregation during meiosis. Results from RNAi-mediated Top1 knockdown lend support to this hypothesis. Our study has provided one of the most comprehensive descriptions of Top1 localization during animal development.


Assuntos
DNA Topoisomerases Tipo I , Drosophila , Animais , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Ribossômico/genética , Drosophila/genética , Drosophila/metabolismo , Meiose , Saccharomyces cerevisiae/genética , Inibidores da Topoisomerase I
7.
Dev Cell ; 56(18): 2623-2635.e5, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34547226

RESUMO

piRNAs guide Piwi/Panoramix-dependent H3K9me3 chromatin modification and transposon silencing during Drosophila germline development. The THO RNA export complex is composed of Hpr1, Tho2, and Thoc5-7. Null thoc7 mutations, which displace Thoc5 and Thoc6 from a Tho2-Hpr1 subcomplex, reduce expression of a subset of germline piRNAs and increase transposon expression, suggesting that THO silences transposons by promoting piRNA biogenesis. Here, we show that the thoc7-null mutant combination increases transposon transcription but does not reduce anti-sense piRNAs targeting half of the transcriptionally activated transposon families. These mutations also fail to reduce piRNA-guided H3K9me3 chromatin modification or block Panoramix-dependent silencing of a reporter transgene, and unspliced transposon transcripts co-precipitate with THO through a Piwi- and Panoramix-independent mechanism. Mutations in piwi also dominantly enhance germline defects associated with thoc7-null alleles. THO thus functions in a piRNA-independent transposon-silencing pathway, which acts cooperatively with Piwi to support germline development.


Assuntos
Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Inativação Gênica/fisiologia , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , Animais , Proteínas Argonauta/genética , Núcleo Celular/metabolismo , Elementos de DNA Transponíveis/genética , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismo
8.
Cells ; 10(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34571985

RESUMO

Golgi phosphoprotein 3 (GOLPH3) is a highly conserved peripheral membrane protein localized to the Golgi apparatus and the cytosol. GOLPH3 binding to Golgi membranes depends on phosphatidylinositol 4-phosphate [PI(4)P] and regulates Golgi architecture and vesicle trafficking. GOLPH3 overexpression has been correlated with poor prognosis in several cancers, but the molecular mechanisms that link GOLPH3 to malignant transformation are poorly understood. We recently showed that PI(4)P-GOLPH3 couples membrane trafficking with contractile ring assembly during cytokinesis in dividing Drosophila spermatocytes. Here, we use affinity purification coupled with mass spectrometry (AP-MS) to identify the protein-protein interaction network (interactome) of Drosophila GOLPH3 in testes. Analysis of the GOLPH3 interactome revealed enrichment for proteins involved in vesicle-mediated trafficking, cell proliferation and cytoskeleton dynamics. In particular, we found that dGOLPH3 interacts with the Drosophila orthologs of Fragile X mental retardation protein and Ataxin-2, suggesting a potential role in the pathophysiology of disorders of the nervous system. Our findings suggest novel molecular targets associated with GOLPH3 that might be relevant for therapeutic intervention in cancers and other human diseases.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Doenças do Sistema Nervoso/metabolismo , Sistema Nervoso/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Proliferação de Células/fisiologia , Citocinese/fisiologia , Citoesqueleto/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Mapas de Interação de Proteínas/fisiologia , Transporte Proteico/fisiologia
9.
Dev Cell ; 56(18): 2664-2680.e6, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34473940

RESUMO

Cachexia, the wasting syndrome commonly observed in advanced cancer patients, accounts for up to one-third of cancer-related mortalities. We have established a Drosophila larval model of organ wasting whereby epithelial overgrowth in eye-antennal discs leads to wasting of the adipose tissue and muscles. The wasting is associated with fat-body remodeling and muscle detachment and is dependent on tumor-secreted matrix metalloproteinase 1 (Mmp1). Mmp1 can both modulate TGFß signaling in the fat body and disrupt basement membrane (BM)/extracellular matrix (ECM) protein localization in both the fat body and the muscle. Inhibition of TGFß signaling or Mmps in the fat body/muscle using a QF2-QUAS binary expression system rescues muscle wasting in the presence of tumor. Altogether, our study proposes that tumor-derived Mmps are central mediators of organ wasting in cancer cachexia.


Assuntos
Tecido Adiposo/metabolismo , Metaloproteinases da Matriz/metabolismo , Músculo Esquelético/metabolismo , Neoplasias/metabolismo , Animais , Membrana Basal/metabolismo , Drosophila/metabolismo , Matriz Extracelular/metabolismo , Atrofia Muscular/metabolismo
10.
Nat Commun ; 12(1): 4987, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404776

RESUMO

In Drosophila, direction-selective neurons implement a mechanism of motion computation similar to cortical neurons, using contrast-opponent receptive fields with ON and OFF subfields. It is not clear how the presynaptic circuitry of direction-selective neurons in the OFF pathway supports this computation if all major inputs are OFF-rectified neurons. Here, we reveal the biological substrate for motion computation in the OFF pathway. Three interneurons, Tm2, Tm9 and CT1, provide information about ON stimuli to the OFF direction-selective neuron T5 across its receptive field, supporting a contrast-opponent receptive field organization. Consistent with its prominent role in motion detection, variability in Tm9 receptive field properties transfers to T5, and calcium decrements in Tm9 in response to ON stimuli persist across behavioral states, while spatial tuning is sharpened by active behavior. Together, our work shows how a key neuronal computation is implemented by its constituent neuronal circuit elements to ensure direction selectivity.


Assuntos
Drosophila/metabolismo , Percepção de Movimento/fisiologia , Movimento (Física) , Neurônios/metabolismo , Animais , Cálcio/metabolismo , Clorfenamidina , Drosophila/genética , Drosophila melanogaster/metabolismo , Feminino , Interneurônios/metabolismo
11.
PLoS Genet ; 17(8): e1009685, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34343194

RESUMO

Gap junction (GJ) proteins, the primary constituents of GJ channels, are conserved determinants of patterning. Canonically, a GJ channel, made up of two hemi-channels contributed by the neighboring cells, facilitates transport of metabolites/ions. Here we demonstrate the involvement of GJ proteins during cuboidal to squamous epithelial transition displayed by the anterior follicle cells (AFCs) from Drosophila ovaries. Somatically derived AFCs stretch and flatten when the adjacent germline cells start increasing in size. GJ proteins, Innexin2 (Inx2) and Innexin4 (Inx4), functioning in the AFCs and germline respectively, promote the shape transformation by modulating calcium levels in the AFCs. Our observations suggest that alterations in calcium flux potentiate STAT activity to influence actomyosin-based cytoskeleton, possibly resulting in disassembly of adherens junctions. Our data have uncovered sequential molecular events underlying the cuboidal to squamous shape transition and offer unique insight into how GJ proteins expressed in the neighboring cells contribute to morphogenetic processes.


Assuntos
Conexinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Células Epiteliais/fisiologia , Folículo Ovariano/fisiologia , Actomiosina/metabolismo , Animais , Padronização Corporal , Sinalização do Cálcio , Conexinas/genética , Citoesqueleto/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Células Epiteliais/metabolismo , Feminino , Morfogênese , Folículo Ovariano/metabolismo
12.
PLoS Genet ; 17(8): e1009709, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34370733

RESUMO

Drosophila larval hematopoiesis occurs in a specialized multi-lobed organ called the lymph gland. Extensive characterization of the organ has provided mechanistic insights into events related to developmental hematopoiesis. Spanning from the thoracic to the abdominal segment of the larvae, this organ comprises a pair of primary, secondary, and tertiary lobes. Much of our understanding arises from the studies on the primary lobe, while the secondary and tertiary lobes have remained mostly unexplored. Previous studies have inferred that these lobes are composed of progenitors that differentiate during pupation; however, the mechanistic basis of this extended progenitor state remains unclear. This study shows that posterior lobe progenitors are maintained by a local signaling center defined by Ubx and Collier in the tertiary lobe. This Ubx zone in the tertiary lobe shares several markers with the niche of the primary lobe. Ubx domain regulates the homeostasis of the posterior lobe progenitors in normal development and an immune-challenged scenario. Our study establishes the lymph gland as a model to tease out how the progenitors interface with the dual niches within an organ during development and disorders.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Hematopoese , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Larva/crescimento & desenvolvimento , Larva/metabolismo , Especificidade de Órgãos , Transdução de Sinais , Nicho de Células-Tronco
13.
Sci Rep ; 11(1): 16791, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408232

RESUMO

Dietary niche is fundamental for determining species ecology; thus, a detailed understanding of what drives variation in dietary niche is vital for predicting ecological shifts and could have implications for species management. Gut microbiota can be important for determining an organism's dietary preference, and therefore which food resources they are likely to exploit. Evidence for whether the composition of the gut microbiota is plastic in response to changes in diet is mixed. Also, the extent to which dietary preference can be changed following colonisation by new gut microbiota from different species is unknown. Here, we use Drosophila spp. to show that: (1) the composition of an individual's gut microbiota can change in response to dietary changes, and (2) ingestion of foreign gut microbes can cause individuals to be attracted to food types they previously had a strong aversion to. Thus, we expose a mechanism for facilitating rapid shifts in dietary niche over short evolutionary timescales.


Assuntos
Dieta , Gorduras Insaturadas na Dieta/farmacologia , Fibras na Dieta/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Animais , Drosophila/metabolismo , Drosophila/microbiologia , Fezes/microbiologia
14.
Sci Rep ; 11(1): 16299, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381067

RESUMO

Correct orchestration of nervous system development is a profound challenge that involves coordination of complex molecular and cellular processes. Mechanistic target of rapamycin (mTOR) signaling is a key regulator of nervous system development and synaptic function. The mTOR kinase is a hub for sensing inputs including growth factor signaling, nutrients and energy levels. Activation of mTOR signaling causes diseases with severe neurological manifestations, such as tuberous sclerosis complex and focal cortical dysplasia. However, the molecular mechanisms by which mTOR signaling regulates nervous system development and function are poorly understood. Unkempt is a conserved zinc finger/RING domain protein that regulates neurogenesis downstream of mTOR signaling in Drosophila. Unkempt also directly interacts with the mTOR complex I component Raptor. Here we describe the generation and characterisation of mice with a conditional knockout of Unkempt (UnkcKO) in the nervous system. Loss of Unkempt reduces Raptor protein levels in the embryonic nervous system but does not affect downstream mTORC1 targets. We also show that nervous system development occurs normally in UnkcKO mice. However, we find that Unkempt is expressed in the adult cerebellum and hippocampus and behavioural analyses show that UnkcKO mice have improved memory formation and cognitive flexibility to re-learn. Further understanding of the role of Unkempt in the nervous system will provide novel mechanistic insight into the role of mTOR signaling in learning and memory.


Assuntos
Cognição/fisiologia , Proteínas de Ligação a DNA/metabolismo , Malformações do Desenvolvimento Cortical/metabolismo , Dedos de Zinco/fisiologia , Animais , Cerebelo/metabolismo , Drosophila/metabolismo , Células HeLa , Hipocampo/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogênese/fisiologia , Transdução de Sinais/fisiologia
15.
J Cell Sci ; 134(17)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34350957

RESUMO

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the endoplasmic reticulum (ER) exit sites under the stress of amino acid starvation. Here, we address which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt-inducible kinases (SIKs; SIK2 and SIK3) by Na+ stress, which, when it is strong, is sufficient. The second is activation of IRE1 and PERK (also known as PEK in flies) downstream of ER stress induced by the absence of amino acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK, and IRE1 and PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work shows the role of SIKs in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino acids and salt. This article has an associated First Person interview with the first author of the paper.


Assuntos
Drosophila , eIF-2 Quinase , Animais , Drosophila/metabolismo , Estresse do Retículo Endoplasmático , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
16.
Development ; 148(18)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34409448

RESUMO

Light-sheet or selective plane illumination microscopy (SPIM) is ideally suited for in toto imaging of living specimens at high temporal-spatial resolution. In SPIM, the light scattering that occurs during imaging of opaque specimens brings about limitations in terms of resolution and the imaging field of view. To ameliorate this shortcoming, the illumination beam can be engineered into a highly confined light sheet over a large field of view and multi-view imaging can be performed by applying multiple lenses combined with mechanical rotation of the sample. Here, we present a Multiview tiling SPIM (MT-SPIM) that combines the Multi-view SPIM (M-SPIM) with a confined, multi-tiled light sheet. The MT-SPIM provides high-resolution, robust and rotation-free imaging of living specimens. We applied the MT-SPIM to image nuclei and Myosin II from the cellular to subcellular spatial scale in early Drosophila embryogenesis. We show that the MT-SPIM improves the axial-resolution relative to the conventional M-SPIM by a factor of two. We further demonstrate that this axial resolution enhancement improves the automated segmentation of Myosin II distribution and of nuclear volumes and shapes.


Assuntos
Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Animais , Drosophila/metabolismo , Drosophila/fisiologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/fisiologia , Miosina Tipo II/metabolismo
17.
Development ; 148(18)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34370012

RESUMO

Drosophila female germline stem cells (GSCs) are found inside the cellular niche at the tip of the ovary. They undergo asymmetric divisions to renew the stem cell lineage and to produce sibling cystoblasts that will in turn enter differentiation. GSCs and cystoblasts contain spectrosomes, membranous structures essential for orientation of the mitotic spindle and that, particularly in GSCs, change shape depending on the cell cycle phase. Using live imaging and a fusion protein of GFP and the spectrosome component Par-1, we follow the complete spectrosome cycle throughout GSC division and quantify the relative duration of the different spectrosome shapes. We also determine that the Par-1 kinase shuttles between the spectrosome and the cytoplasm during mitosis and observe the continuous addition of new material to the GSC and cystoblast spectrosomes. Next, we use the Fly-FUCCI tool to define, in live and fixed tissues, that GSCs have a shorter G1 compared with the G2 phase. The observation of centrosomes in dividing GSCs allowed us to determine that centrosomes separate very early in G1, before centriole duplication. Furthermore, we show that the anterior centrosome associates with the spectrosome only during mitosis and that, upon mitotic spindle assembly, it translocates to the cell cortex, where it remains anchored until centrosome separation. Finally, we demonstrate that the asymmetric division of GSCs is not an intrinsic property of these cells, as the spectrosome of GSC-like cells located outside of the niche can divide symmetrically. Thus, GSCs display unique properties during division, a behaviour influenced by the surrounding niche.


Assuntos
Divisão Celular Assimétrica/fisiologia , Centrossomo/fisiologia , Drosophila/fisiologia , Células Germinativas/fisiologia , Ovário/fisiologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular/fisiologia , Centrossomo/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Feminino , Fase G1/fisiologia , Fase G2/fisiologia , Células Germinativas/metabolismo , Mitose/fisiologia , Ovário/metabolismo , Fuso Acromático/fisiologia , Células-Tronco/metabolismo
18.
Development ; 148(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34355743

RESUMO

Because both dearth and overabundance of histones result in cellular defects, histone synthesis and demand are typically tightly coupled. In Drosophila embryos, histones H2B, H2A and H2Av accumulate on lipid droplets (LDs), which are cytoplasmic fat storage organelles. Without LD binding, maternally provided H2B, H2A and H2Av are absent; however, how LDs ensure histone storage is unclear. Using quantitative imaging, we uncover when during oogenesis these histones accumulate, and which step of accumulation is LD dependent. LDs originate in nurse cells (NCs) and are transported to the oocyte. Although H2Av accumulates on LDs in NCs, the majority of the final H2Av pool is synthesized in oocytes. LDs promote intercellular transport of the histone anchor Jabba and thus its presence in the ooplasm. Ooplasmic Jabba then prevents H2Av degradation, safeguarding the H2Av stockpile. Our findings provide insight into the mechanism for establishing histone stores during Drosophila oogenesis and shed light on the function of LDs as protein-sequestration sites.


Assuntos
Histonas/metabolismo , Gotículas Lipídicas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Oócitos/metabolismo , Oogênese/fisiologia
19.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445636

RESUMO

The Drosophila eye has been used extensively to study numerous aspects of biological systems, for example, spatio-temporal regulation of differentiation, visual signal transduction, protein trafficking and neurodegeneration. Right from the advent of fluorescent proteins (FPs) near the end of the millennium, heterologously expressed fusion proteins comprising FPs have been applied in Drosophila vision research not only for subcellular localization of proteins but also for genetic screens and analysis of photoreceptor function. Here, we summarize applications for FPs used in the Drosophila eye as part of genetic screens, to study rhodopsin expression patterns, subcellular protein localization, membrane protein transport or as genetically encoded biosensors for Ca2+ and phospholipids in vivo. We also discuss recently developed FPs that are suitable for super-resolution or correlative light and electron microscopy (CLEM) approaches. Illustrating the possibilities provided by using FPs in Drosophila photoreceptors may aid research in other sensory or neuronal systems that have not yet been studied as well as the Drosophila eye.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas Luminescentes/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Vias Visuais/metabolismo , Animais , Transporte Proteico
20.
Cell Mol Life Sci ; 78(19-20): 6593-6603, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34448011

RESUMO

The concept of reverse chemical ecology (exploitation of molecular knowledge for chemical ecology) has recently emerged in conservation biology and human health. Here, we extend this concept to crop protection. Targeting odorant receptors from a crop pest insect, the noctuid moth Spodoptera littoralis, we demonstrate that reverse chemical ecology has the potential to accelerate the discovery of novel crop pest insect attractants and repellents. Using machine learning, we first predicted novel natural ligands for two odorant receptors, SlitOR24 and 25. Then, electrophysiological validation proved in silico predictions to be highly sensitive, as 93% and 67% of predicted agonists triggered a response in Drosophila olfactory neurons expressing SlitOR24 and SlitOR25, respectively, despite a lack of specificity. Last, when tested in Y-maze behavioral assays, the most active novel ligands of the receptors were attractive to caterpillars. This work provides a template for rational design of new eco-friendly semiochemicals to manage crop pest populations.


Assuntos
Mariposas/efeitos dos fármacos , Mariposas/metabolismo , Receptores Odorantes/metabolismo , Animais , Drosophila/efeitos dos fármacos , Drosophila/metabolismo , Proteínas de Insetos/metabolismo , Repelentes de Insetos/farmacologia , Aprendizado de Máquina , Odorantes , Feromônios/farmacologia , Olfato/efeitos dos fármacos , Spodoptera/efeitos dos fármacos , Spodoptera/metabolismo
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