RESUMEN
How canonical cytokinesis is altered during germ cell division to produce stable intercellular bridges, called "ring canals," is poorly understood. Here, using time-lapse imaging in Drosophila, we observe that ring canal formation occurs through extensive remodeling of the germ cell midbody, a structure classically associated with its function in recruiting abscission-regulating proteins in complete cytokinesis. Germ cell midbody cores reorganize and join the midbody ring rather than being discarded, and this transition is accompanied by changes in centralspindlin dynamics. The midbody-to-ring canal transformation is conserved in the Drosophila male and female germlines and during mouse and Hydra spermatogenesis. In Drosophila, ring canal formation depends on Citron kinase function to stabilize the midbody, similar to its role during somatic cell cytokinesis. Our results provide important insights into the broader functions of incomplete cytokinesis events across biological systems, such as those observed during development and disease states.
Asunto(s)
Citocinesis , Espermatogénesis , Masculino , Animales , Ratones , Citocinesis/fisiología , División Celular , Células Germinativas , DrosophilaRESUMEN
Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.
Asunto(s)
Microscopía Fluorescente/métodos , Nanotecnología/métodos , Óptica y Fotónica/métodos , Imagenología Tridimensional , Relación Señal-RuidoRESUMEN
Translational stop codon readthrough occurs in organisms ranging from viruses to mammals and is especially prevalent in decoding Drosophila and viral mRNAs. Recoding of UGA, UAG, or UAA to specify an amino acid allows a proportion of the protein encoded by a single gene to be C-terminally extended. The extended product from Drosophila kelch mRNA is 160 kDa, whereas unextended Kelch protein, a subunit of a Cullin3-RING ubiquitin ligase, is 76 kDa. Previously we reported tissue-specific regulation of readthrough of the first kelch stop codon. Here, we characterize major efficiency differences in a variety of cell types. Immunoblotting revealed low levels of readthrough in malpighian tubules, ovary, and testis but abundant readthrough product in lysates of larval and adult central nervous system (CNS) tissue. Reporters of readthrough demonstrated greater than 30% readthrough in adult brains, and imaging in larval and adult brains showed that readthrough occurred in neurons but not glia. The extent of readthrough stimulatory sequences flanking the readthrough stop codon was assessed in transgenic Drosophila and in human tissue culture cells where inefficient readthrough occurs. A 99-nucleotide sequence with potential to form an mRNA stem-loop 3' of the readthrough stop codon stimulated readthrough efficiency. However, even with just six nucleotides of kelch mRNA sequence 3' of the stop codon, readthrough efficiency only dropped to 6% in adult neurons. Finally, we show that high-efficiency readthrough in the Drosophila CNS is common; for many neuronal proteins, C-terminal extended forms of individual proteins are likely relatively abundant.
Asunto(s)
Codón/genética , Drosophila melanogaster/genética , Especificidad de Órganos/genética , Animales , Sistema Nervioso Central/metabolismo , ADN Complementario/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Genes Reporteros , Células HEK293 , Humanos , Discos Imaginales/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Animal germ cells communicate directly with each other during gametogenesis through intercellular bridges, often called ring canals (RCs), that form as a consequence of incomplete cytokinesis during cell division. Developing germ cells in Drosophila have an additional specialized organelle connecting the cells called the fusome. Ring canals and the fusome are required for fertility in Drosophila females, but little is known about their roles during spermatogenesis. With live imaging, we directly observe the intercellular movement of GFP and a subset of endogenous proteins through RCs during spermatogenesis, from two-cell diploid spermatogonia to clusters of 64 post-meiotic haploid spermatids, demonstrating that RCs are stable and open to intercellular traffic throughout spermatogenesis. Disruption of the fusome, a large cytoplasmic structure that extends through RCs and is important during oogenesis, had no effect on spermatogenesis or male fertility under normal conditions. Our results reveal that male germline RCs allow the sharing of cytoplasmic information that might play a role in quality control surveillance during sperm development.
Asunto(s)
Citoplasma/metabolismo , Meiosis/fisiología , Espermátides/metabolismo , Espermatogénesis/fisiología , Espermatogonias/metabolismo , Animales , Citoplasma/genética , Drosophila melanogaster , Masculino , Espermátides/citología , Espermatogonias/citologíaRESUMEN
Ring canals in the female germline of Drosophila melanogaster are supported by a robust filamentous actin (F-actin) cytoskeleton, setting them apart from ring canals in other species and tissues. Previous work has identified components required for the expansion of the ring canal actin cytoskeleton, but has not identified the proteins responsible for F-actin recruitment or accumulation. Using a combination of CRISPR-Cas9 mediated mutagenesis and UAS-Gal4 overexpression, we show that HtsRC-a component specific to female germline ring canals-is both necessary and sufficient to drive F-actin accumulation. Absence of HtsRC in the germline resulted in ring canals lacking inner rim F-actin, while overexpression of HtsRC led to larger ring canals. HtsRC functions in combination with Filamin to recruit F-actin to ectopic actin structures in somatic follicle cells. Finally, we present findings that indicate that HtsRC expression and robust female germline ring canal expansion are important for high fecundity in fruit flies but dispensable for their fertility-a result that is consistent with our understanding of HtsRC as a newly evolved gene specific to female germline ring canals.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Citocinesis , Proteínas de Drosophila/metabolismo , Oogénesis , Actinas/metabolismo , Animales , Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Femenino , Filaminas/metabolismo , Óvulo/citología , Óvulo/metabolismoRESUMEN
Gametogenesis is dependent on intercellular communication facilitated by stable intercellular bridges connecting developing germ cells. During Drosophila oogenesis, intercellular bridges (referred to as ring canals; RCs) have a dynamic actin cytoskeleton that drives their expansion to a diameter of 10â µm. Although multiple proteins have been identified as components of RCs, we lack a basic understanding of how RC proteins interact together to form and regulate the RC cytoskeleton. Thus, here, we optimized a procedure for proximity-dependent biotinylation in live tissue using the APEX enzyme to interrogate the RC interactome. APEX was fused to four different RC components (RC-APEX baits) and 55 unique high-confidence prey were identified. The RC-APEX baits produced almost entirely distinct interactomes that included both known RC proteins and uncharacterized proteins. A proximity ligation assay was used to validate close-proximity interactions between the RC-APEX baits and their respective prey. Furthermore, an RNA interference screen revealed functional roles for several high-confidence prey genes in RC biology. These findings highlight the utility of enzyme-catalyzed proximity labeling for protein interactome analysis in live tissue and expand our understanding of RC biology.
Asunto(s)
Comunicación Celular/genética , Células Germinativas/metabolismo , Imagen Molecular/métodos , Oogénesis , Mapas de Interacción de Proteínas/fisiología , Coloración y Etiquetado/métodos , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Diferenciación Celular/genética , Técnicas Citológicas/métodos , Citoesqueleto/genética , Citoesqueleto/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Drosophila melanogaster/genética , Femenino , Genes Reporteros , Uniones Intercelulares/fisiología , Oocitos/metabolismo , Oogénesis/genética , Unión Proteica , Mapas de Interacción de Proteínas/genéticaRESUMEN
During Drosophila oogenesis, specialized actin-based structures called ring canals form and expand to accommodate growth of the oocyte. Previous work demonstrated that Kelch and Cullin 3 function together in a Cullin 3-RING ubiquitin ligase complex (CRL3Kelch) to organize the ring canal cytoskeleton, presumably by targeting a substrate for proteolysis. Here, we use tandem affinity purification followed by mass spectrometry to identify HtsRC as the CRL3Kelch ring canal substrate. CRISPR-mediated mutagenesis of HtsRC revealed its requirement in the recruitment of the ring canal F-actin cytoskeleton. We present genetic evidence consistent with HtsRC being the CRL3Kelch substrate, as well as biochemical evidence indicating that HtsRC is ubiquitylated and degraded by the proteasome. Finally, we identify a short sequence motif in HtsRC that is necessary for Kelch binding. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitin-proteasome system.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Microfilamentos/metabolismo , Oocitos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitinación , Citoesqueleto de Actina/genética , Actinas/genética , Actinas/metabolismo , Animales , Sistemas CRISPR-Cas , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas de Microfilamentos/genética , Mutagénesis , Oocitos/citología , Complejo de la Endopetidasa Proteasomal/genéticaRESUMEN
The Germ Cell-Less (GCL) protein is a key regulator of primordial germ cell (PGC) formation in Drosophila embryos. Reporting in this issue of Developmental Cell, Pae et al. (2017) show that GCL blocks somatic cell fate by specifically destroying the Torso Receptor Tyrosine Kinase.
Asunto(s)
Proteínas de Drosophila , Células Germinativas , Animales , Drosophila , Proteínas Tirosina Quinasas ReceptorasRESUMEN
The Drosophila Kelch protein is required to organize the ovarian ring canal cytoskeleton. Kelch binds and cross-links F-actin in vitro, and it also functions with Cullin 3 (Cul3) as a component of a ubiquitin E3 ligase. How these two activities contribute to cytoskeletal remodeling in vivo is not known. We used targeted mutagenesis to investigate the mechanism of Kelch function. We tested a model in which Cul3-dependent degradation of Kelch is required for its function, but we found no evidence to support this hypothesis. However, we found that mutant Kelch deficient in its ability to interact with Cul3 failed to rescue the kelch cytoskeletal defects, suggesting that ubiquitin ligase activity is the principal activity required in vivo. We also determined that the proteasome is required with Kelch to promote the ordered growth of the ring canal cytoskeleton. These results indicate that Kelch organizes the cytoskeleton in vivo by targeting a protein substrate for degradation by the proteasome.
Asunto(s)
Actinas/fisiología , Proteínas Cullin/fisiología , Proteínas de Drosophila/fisiología , Proteínas de Microfilamentos/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Citoesqueleto/fisiología , Drosophila/anatomía & histología , Drosophila/citología , Proteínas de Drosophila/genética , Femenino , Genitales Femeninos/citología , Proteínas de Microfilamentos/genética , Mutagénesis , Fenotipo , Complejo de la Endopetidasa Proteasomal/fisiología , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/químicaRESUMEN
To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).
Asunto(s)
Drosophila/genética , Interferencia de ARN , Acceso a la Información , Animales , Animales Modificados Genéticamente , Investigación Biomédica , Boston , Genes de Insecto , Vectores Genéticos , Facultades de MedicinaRESUMEN
Egg chambers from starved Drosophila females contain large aggregates of processing (P) bodies and cortically enriched microtubules. As this response to starvation is rapidly reversed upon re-feeding females or culturing egg chambers with exogenous bovine insulin, we examined the role of endogenous insulin signaling in mediating the starvation response. We found that systemic Drosophila insulin-like peptides (dILPs) activate the insulin pathway in follicle cells, which then regulate both microtubule and P body organization in the underlying germline cells. This organization is modulated by the motor proteins Dynein and Kinesin. Dynein activity is required for microtubule and P body organization during starvation, while Kinesin activity is required during nutrient-rich conditions. Blocking the ability of egg chambers to form P body aggregates in response to starvation correlated with reduced progeny survival. These data suggest a potential mechanism to maximize fecundity even during periods of poor nutrient availability, by mounting a protective response in immature egg chambers.
Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Alimentos , Células Germinativas/metabolismo , Insulina/metabolismo , Óvulo/citología , Transducción de Señal , Animales , Apoptosis , Bovinos , Estructuras Citoplasmáticas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Embrión no Mamífero/metabolismo , Femenino , Microtúbulos/metabolismo , Modelos Biológicos , Oocitos/metabolismo , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , Óvulo/metabolismo , Péptidos/metabolismo , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an antivirulence agent against diverse bacteria, including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest therapeutic strategies for countering pathogens.
Asunto(s)
Antibacterianos/farmacología , Proteínas Anticongelantes/farmacología , Proteínas de Insectos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Animales , Proteínas Anticongelantes/genética , Biopelículas/efectos de los fármacos , Resistencia a la Enfermedad/genética , Drosophila/genética , Drosophila/microbiología , Proteínas de Insectos/genética , Ixodes/química , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/inmunologíaRESUMEN
Stem cells possess the capacity to generate two cells of distinct fate upon division: one cell retaining stem cell identity and the other cell destined to differentiate. These cell fates are established by cell-type-specific genetic networks. To comprehensively identify components of these networks, we performed a large-scale RNAi screen in Drosophila female germline stem cells (GSCs) covering â¼25% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation, or other processes involved in oogenesis. Comparison of GSC regulators with neural stem cell self-renewal factors identifies common and cell-type-specific self-renewal genes. Importantly, we identify the histone methyltransferase Set1 as a GSC-specific self-renewal factor. Loss of Set1 in neural stem cells does not affect cell fate decisions, suggesting a differential requirement of H3K4me3 in different stem cell lineages. Altogether, our study provides a resource that will help to further dissect the networks underlying stem cell self-renewal.
Asunto(s)
Diferenciación Celular , División Celular/fisiología , Linaje de la Célula/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Germinativas/citología , Células Madre/citología , Animales , Diferenciación Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Femenino , Células Germinativas/metabolismo , Ovario/citología , Ovario/metabolismo , Interferencia de ARN/fisiología , Transducción de Señal/fisiología , Células Madre/metabolismoRESUMEN
Drosophila oogenesis is an excellent system for the study of developmental cell biology. Active areas of research include stem cell maintenance, gamete development, pattern formation, cytoskeletal regulation, intercellular communication, intercellular transport, cell polarity, cell migration, cell death, morphogenesis, cell cycle control, and many more. The large size and relatively simple organization of egg chambers make them ideally suited for microscopy of both living and fixed whole mount tissue. A wide range of tools is available for oogenesis research. Newly available shRNA transgenic lines provide an alternative to classic loss-of-function F2 screens and clonal screens. Gene expression can be specifically controlled in either germline or somatic cells using the Gal4/UAS system. Protein trap lines provide fluorescent tags of proteins expressed at endogenous levels for live imaging and screening backgrounds. This review provides information on many available reagents and key methods for research in oogenesis.
Asunto(s)
Diferenciación Celular/genética , Oogénesis/genética , Transcripción Genética , Animales , Movimiento Celular/genética , Biología Evolutiva/métodos , Drosophila , Femenino , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Ring canals are made from arrested cleavage furrows, and provide direct cytoplasmic connections among sibling cells. They are well documented for their participation in Drosophila oogenesis, but little is known about their role in several somatic tissues in which they are also found. Using a variety of genetic tools in live and fixed tissue, we recently demonstrated that rapid intercellular exchange occurs through somatic ring canals by diffusion, and presented evidence that ring canals permit equilibration of protein among transcriptionally mosaic cells. We also used a novel combination of markers to evaluate the extent of protein movement within and across mitotic clones in follicle cells and imaginal discs, providing evidence of robust movement of GFP between the 2 sides of mitotic clones and frequently into non-recombined cells. These data suggest that, depending on the experimental setup and proteins of interest, inter-clonal diffusion of protein may alter the interpretation of clonal data in follicle cells. Here, we discuss these results and provide additional insight into the impact of ring canals in Drosophila somatic tissues.
Asunto(s)
Proteínas de Drosophila/metabolismo , Uniones Intercelulares/fisiología , Animales , Drosophila , Femenino , Células Gigantes/citología , MasculinoRESUMEN
The application of two-photon activation of photoactivatable fluorescent proteins is limited by a lack of information about two-photon activation rates. Here we present rates for the commonly used photoactivatable proteins PAmCherry, PAmKate and PA-GFP at different wavelengths using a novel method that allows us to determine the two-photon activation rates directly, independent of any reference data, with microscopic sample volumes. We show that PAmCherry features the highest rates of the tested proteins at 700 nm activation wavelength followed by PAmKate. Towards longer wavelengths, two-photon activation rates decrease for all three proteins. For PAmCherry, our data contradicts an activation model relying solely on two-photon activation and suggests additional activation pathways requiring at least two absorption steps. Our method is readily expandable to other photoactivatable fluorescent molecules. The presented results allow optimization of experimental conditions in spectroscopic and imaging techniques such as super-resolution fluorescence microscopy.
Asunto(s)
Proteínas Luminiscentes/química , Fotones , Bacterias/química , Bacterias/citología , Células Cultivadas , Procesos FotoquímicosRESUMEN
Although intercellular bridges resulting from incomplete cytokinesis were discovered in somatic Drosophila tissues decades ago, the impact of these structures on intercellular communication and tissue biology is largely unknown. In this work, we demonstrate that the ~250-nanometer-diameter somatic ring canals permit diffusion of cytoplasmic contents between connected cells and across mitotic clone boundaries and enable the equilibration of protein between transcriptionally mosaic follicle cells in the Drosophila ovary. We obtained similar, although more restricted, results in the larval imaginal discs. Our work illustrates the lack of cytoplasmic autonomy in these tissues and suggests a role for somatic ring canals in promoting homogeneous protein expression within the tissue.
Asunto(s)
Citoplasma/metabolismo , Estructuras Citoplasmáticas/metabolismo , Estructuras Citoplasmáticas/ultraestructura , Proteínas de Drosophila/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Discos Imaginales/metabolismo , Discos Imaginales/ultraestructura , Transporte de Proteínas , Animales , Ciclo Celular , Difusión , Drosophila , Femenino , Células Gigantes/ultraestructura , Microscopía Electrónica , Mitosis , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , Folículo Ovárico/ultraestructura , Recombinación Genética , Transcripción Genética , TransgenesRESUMEN
Drosophila melanogaster experience cold shock injury and die when exposed to low non-freezing temperatures. In this study, we generated transgenic D. melanogaster that express putative Ixodes scapularis antifreeze glycoprotein (IAFGP) and show that the presence of IAFGP increases the ability of flies to survive in the cold. Male and female adult iafgp-expressing D. melanogaster exhibited higher survival rates compared with controls when placed at non-freezing temperatures. Increased hatching rates were evident in embryos expressing IAFGP when exposed to the cold. The TUNEL assay showed that flight muscles from iafgp-expressing female adult flies exhibited less apoptotic damage upon exposure to non-freezing temperatures in comparison to control flies. Collectively, these data suggest that expression of iafgp increases cold tolerance in flies by preventing apoptosis. This study defines a molecular basis for the role of an antifreeze protein in cryoprotection of flies.
Asunto(s)
Aclimatación/efectos de los fármacos , Proteínas Anticongelantes/metabolismo , Apoptosis/efectos de los fármacos , Frío , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiología , Ixodes/química , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Proteínas Anticongelantes/genética , Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , Embrión no Mamífero/fisiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Immunoblotting , Etiquetado Corte-Fin in Situ , Masculino , Músculos/metabolismo , Oligonucleótidos/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Ring canals connecting Drosophila germline, follicle and imaginal disc cells provide direct contact of cytoplasm between cells. To date, little is known about the formation, structure, or function of the somatic ring canals present in follicle and imaginal disc cells. Here, we show by confocal and electron microscopy that Pavarotti kinesin-like protein and Visgun are stable components of somatic ring canals. Using live-cell confocal microscopy, we show that somatic ring canals form from the stabilization of mitotic cleavage furrows. In contrast to germline cells, syncytial follicle cells do not divide synchronously, are not maximally branched and their ring canals do not increase in size during egg chamber development. We show for the first time that somatic ring canals permit exchange of cytoplasmic proteins between follicle cells. These results provide insight into the composition and function of ring canals in somatic cells, implying a broader functional significance for syncytial organization of cells outside the germline.