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1.
Curr Biol ; 33(17): 3747-3758.e9, 2023 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-37552984

RESUMO

Cilia allowed our protistan ancestors to sense and explore their environment, avoid predation, and capture bacterial prey.1,2,3 Regulated ciliogenesis was likely critical for early animal evolution,2,4,5,6 and in modern animals, deploying cilia in the right cells at the right time is crucial for development and physiology. Two transcription factors, RFX and FoxJ1, coordinate ciliogenesis in animals7,8,9 but are absent from the genomes of many other ciliated eukaryotes, raising the question of how the regulation of ciliogenesis in animals evolved.10,11 By comparing the genomes of animals with those of their closest living relatives, the choanoflagellates, we found that the genome of their last common ancestor encoded at least three RFX paralogs and a FoxJ1 homolog. Disruption of the RFX homolog cRFXa in the model choanoflagellate Salpingoeca rosetta resulted in delayed cell proliferation and aberrant ciliogenesis, marked by the collapse and resorption of nascent cilia. In cRFXa mutants, ciliogenesis genes and foxJ1 were significantly downregulated. Moreover, the promoters of S. rosetta ciliary genes are enriched for DNA motifs matching those bound by the cRFXa protein in vitro. These findings suggest that an ancestral cRFXa homolog coordinated ciliogenesis in the progenitors of animals and choanoflagellates and that the selective deployment of the RFX regulatory module may have been necessary to differentiate ciliated from non-ciliated cell types during early animal evolution.


Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição , Animais , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição de Fator Regulador X/genética , Fatores de Transcrição de Fator Regulador X/metabolismo , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Cílios/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo
2.
Proc Natl Acad Sci U S A ; 120(28): e2302445120, 2023 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-37399378

RESUMO

Cells regulate gene expression by the specific binding of transcription regulators to cis-regulatory sequences. Pair-wise cooperativity between regulators-whereby two different regulators physically interact and bind DNA in a cooperative manner-is common and permits complex modes of gene regulation. Over evolutionary timescales, the formation of new combinations of regulators represents a major source of phenotypic novelty, facilitating new network structures. How functional, pair-wise cooperative interactions arise between regulators is poorly understood, despite the abundance of examples in extant species. Here, we explore a protein-protein interaction between two ancient transcriptional regulators-the homeodomain protein Matα2 and the MADS box protein Mcm1-that was gained approximately 200 million y ago in a clade of ascomycete yeasts that includes Saccharomyces cerevisiae. By combining deep mutational scanning with a functional selection for cooperative gene expression, we tested millions of possible alternative evolutionary solutions to this interaction interface. The artificially evolved, functional solutions are highly degenerate, with diverse amino acid chemistries permitted at all positions but with widespread epistasis limiting success. Nonetheless, approximately ~45% of the random sequences sampled function as well or better in controlling gene expression than the naturally evolved sequence. From these variants (which are unconstrained by historical contingency), we discern structural rules and epistatic constraints governing the emergence of cooperativity between these two transcriptional regulators. This work provides a mechanistic basis for long-standing observations of transcription network plasticity and highlights the importance of epistasis in the evolution of new protein-protein interactions.


Assuntos
Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regulação da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
PLoS One ; 13(10): e0201865, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30365488

RESUMO

Aphids, like most animals, mount a diverse set of defenses against pathogens. For aphids, two of the best studied defenses are symbiont-conferred protection and transgenerational wing induction. Aphids can harbor bacterial symbionts that provide protection against pathogens, parasitoids and predators, as well as against other environmental stressors. In response to signals of danger, aphids also protect not themselves but their offspring by producing more winged than unwinged offspring as a way to ensure that their progeny may be able to escape deteriorating conditions. Such transgenerational wing induction has been studied most commonly as a response to overcrowding of host plants and presence of predators, but recent evidence suggests that pea aphids (Acyrthosiphon pisum) may also begin to produce a greater proportion of winged offspring when infected with fungal pathogens. Here, we explore this phenomenon further by asking how protective symbionts, pathogen dosage and environmental conditions influence this response. Overall, while we find some evidence that protective symbionts can modulate transgenerational wing induction in response to fungal pathogens, we observe that transgenerational wing induction in response to fungal infection is highly variable. That variability cannot be explained entirely by symbiont association, by pathogen load or by environmental stress, leaving the possibility that a complex interplay of genotypic and environmental factors may together influence this trait.


Assuntos
Afídeos/genética , Ecologia , Micoses/genética , Simbiose/genética , Animais , Afídeos/crescimento & desenvolvimento , Afídeos/microbiologia , Fungos/patogenicidade , Micoses/microbiologia , Fenótipo , Simbiose/fisiologia , Vespas/genética , Vespas/crescimento & desenvolvimento , Vespas/microbiologia , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/microbiologia
4.
Viruses ; 10(8)2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30127286

RESUMO

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 µm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.


Assuntos
Vírus Sincicial Respiratório Humano/ultraestrutura , Vírion/ultraestrutura , Montagem de Vírus/fisiologia , Células A549 , Animais , Brônquios/virologia , Linhagem Celular , Chlorocebus aethiops , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Células Epiteliais/ultraestrutura , Células Epiteliais/virologia , Células HeLa , Humanos , Microtomia , Vírus Sincicial Respiratório Humano/fisiologia , Células Vero , Vírion/fisiologia
5.
Nat Commun ; 9(1): 1736, 2018 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-29712906

RESUMO

Measles virus (MeV) remains a major human pathogen, but there are presently no licensed antivirals to treat MeV or other paramyxoviruses. Here, we use cryo-electron tomography (cryo-ET) to elucidate the principles governing paramyxovirus assembly in MeV-infected human cells. The three-dimensional (3D) arrangement of the MeV structural proteins including the surface glycoproteins (F and H), matrix protein (M), and the ribonucleoprotein complex (RNP) are characterized at stages of virus assembly and budding, and in released virus particles. The M protein is observed as an organized two-dimensional (2D) paracrystalline array associated with the membrane. A two-layered F-M lattice is revealed suggesting that interactions between F and M may coordinate processes essential for MeV assembly. The RNP complex remains associated with and in close proximity to the M lattice. In this model, the M lattice facilitates the well-ordered incorporation and concentration of the surface glycoproteins and the RNP at sites of virus assembly.


Assuntos
Hemaglutininas Virais/ultraestrutura , Vírus do Sarampo/ultraestrutura , Ribonucleoproteínas/ultraestrutura , Proteínas Virais de Fusão/ultraestrutura , Proteínas da Matriz Viral/ultraestrutura , Vírion/ultraestrutura , Linhagem Celular , Microscopia Crioeletrônica , Fibroblastos/ultraestrutura , Fibroblastos/virologia , Células HeLa , Hemaglutininas Virais/metabolismo , Humanos , Vírus do Sarampo/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais de Fusão/metabolismo , Proteínas da Matriz Viral/metabolismo , Vírion/metabolismo , Montagem de Vírus , Liberação de Vírus
6.
Nat Protoc ; 12(1): 150-167, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27977021

RESUMO

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5-15 d for an individual experienced in cryo-EM.


Assuntos
Microscopia Crioeletrônica/métodos , HIV-1/fisiologia , Herpesvirus Humano 1/fisiologia , Microscopia de Fluorescência/métodos , Transfecção , Linhagem Celular , Humanos
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