RESUMEN
Bats stand out among mammalian species for their exceptional traits, including the capacity to navigate through flight and echolocation, conserve energy through torpor/hibernation, harbor a multitude of viruses, exhibit resistance to disease, survive harsh environmental conditions, and demonstrate exceptional longevity compared to other mammals of similar size. In vivo studies of bats are challenging for several reasons, such as difficulty in locating and capturing them in their natural environments, limited accessibility, low sample size, environmental variation, long lifespans, slow reproductive rates, zoonotic disease risks, species protection, and ethical concerns. Thus, establishing alternative laboratory models is crucial for investigating the diverse physiological adaptations observed in bats. Obtaining quality cells from tissues is a critical first step for successful primary cell derivation. However, it is often impractical to collect fresh tissue and process the samples immediately for cell culture due to the resources required for isolating and expanding cells. As a result, frozen tissue is typically the starting resource for bat primary cell derivation, but cells in frozen tissue are usually damaged and have low integrity and viability. Isolating primary cells from frozen tissues thus poses a significant challenge. Herein, we present a successfully developed protocol for isolating primary dermal fibroblasts from frozen bat wing biopsies. This protocol marks a significant milestone, as this is the first protocol specifically focused on fibroblast isolation from bat frozen tissue. We also describe methods for primary cell characterization, genetic manipulation of primary cells through lentivirus transduction, and the development of stable cell lines. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Bat wing biopsy collection and preservation Support Protocol 1: Blood collection from bat venipuncture Basic Protocol 2: Isolation of primary fibroblasts from adult bat frozen wing biopsy Support Protocol 2: Primary fibroblast culture and subculture Support Protocol 3: Determination of growth curve and doubling time Support Protocol 4: Cell banking and thawing of primary fibroblasts Basic Protocol 3: Lentiviral transduction of bat primary fibroblasts Basic Protocol 4: Bat stable fibroblast cell line development Support Protocol 5: Bat fibroblast validation by immunofluorescence staining Basic Protocol 5: Chromosome counting.
Asunto(s)
Quirópteros , Criopreservación , Fibroblastos , Alas de Animales , Animales , Quirópteros/virología , Fibroblastos/citología , Criopreservación/métodos , Línea Celular , Biopsia/métodos , Técnicas de Cultivo de Célula/métodos , CongelaciónRESUMEN
Dietary specializations in animals lead to adaptations in morphology, anatomy and physiology. Neotropical bats, with their high taxonomic and trophic diversity, offer a unique perspective on diet-driven evolutionary adaptations. Here we assess the metabolic response to different dietary sugars among wild-caught bats. We found that insectivorous bats had a pronounced metabolic response to trehalose, whereas bats with nectar and fruit-based diets showed significantly higher blood glucose levels in response to glucose and sucrose, reaching levels over 750 mg dl-1. The genomic analysis of 22 focal species and two outgroup species identified positive selection for the digestive enzyme trehalase in insect eaters, while sucrase-isomaltase showed selection in lineages with omnivorous and nectar diets. By examining anatomical and cellular features of the small intestine, we discovered that dietary sugar proportion strongly impacted numerous digestive traits, providing valuable insight into the physiological implications of molecular adaptations. Using hybridization chain reaction (HCR) RNA fluorescence in situ hybridization, we observed unusually high expression in the glucose transporter gene Slc2a2 in nectar bats, while fruit bats increased levels of Slc5a1 and Slc2a5. Overall, this study highlights the intricate interplay between molecular, morphological and physiological aspects of diet evolution, offering new insights into the mechanisms of dietary diversification and sugar assimilation in mammals.
Asunto(s)
Evolución Biológica , Quirópteros , Dieta , Animales , Quirópteros/fisiología , Quirópteros/genética , Dieta/veterinaria , Azúcares de la DietaRESUMEN
Bats stand out among mammalian species for their exceptional traits, including the capacity to navigate through flight and echolocation, conserve energy through torpor/hibernation, harbor a multitude of viruses, exhibit resistance to disease, survive harsh environmental conditions, and demonstrate exceptional longevity compared to other mammals of similar size. In vivo studies of bats can be challenging for several reasons such as ability to locate and capture them in their natural environments, limited accessibility, low sample size, environmental variation, long lifespans, slow reproductive rates, zoonotic disease risks, species protection, and ethical concerns. Thus, establishing alternative laboratory models is crucial for investigating the diverse physiological adaptations observed in bats. Obtaining quality cells from tissues is a critical first step for successful primary cell derivation. However, it is often impractical to collect fresh tissue and process the samples immediately for cell culture due to the resources required for isolating and expanding cells. As a result, frozen tissue is typically the starting resource for bat primary cell derivation. Yet, cells in frozen tissue are usually damaged and represent low integrity and viability. As a result, isolating primary cells from frozen tissues poses a significant challenge. Herein, we present a successfully developed protocol for isolating primary dermal fibroblasts from frozen bat wing biopsies. This protocol marks a significant milestone, as this the first protocol specially focused on fibroblasts isolation from bat frozen tissue. We also describe methods for primary cell characterization, genetic manipulation of primary cells through lentivirus transduction, and the development of stable cell lines. Basic Protocol 1: Bat wing biopsy collection and preservation Support Protocol 1: Blood collection from bat- venipuncture Basic Protocol 2: Isolation of primary fibroblasts from adult bat frozen wing biopsy Support Protocol 2: Maintenance of primary fibroblasts Support Protocol 3: Cell banking and thawing of primary fibroblasts Support Protocol 4: Growth curve and doubling time Support Protocol 5: Lentiviral transduction of bat primary fibroblasts Basic Protocol 3: Bat stable fibroblasts cell lines development Support Protocol 6: Bat fibroblasts validation by immunofluorescence staining Support Protocol 7: Chromosome counting.
RESUMEN
The Spt-Ada-Gcn5 Acetyltransferase (SAGA) chromatin modifying complex is a critical regulator of gene expression and is highly conserved across species. Subunits of SAGA arrange into discrete modules with lysine aceyltransferase and deubiquitinase activities housed separately. Mutation of the SAGA deubiquitinase module can lead to substantial biological misfunction and diseases such as cancer, neurodegeneration, and blindness. Here, we review the structure and functions of the SAGA deubiquitinase module and regulatory mechanisms acting to control these.
Asunto(s)
Enzimas Desubicuitinizantes/metabolismo , Complejos Multienzimáticos/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Factores de Transcripción p300-CBP/metabolismo , Animales , Arabidopsis/enzimología , Aspergillus nidulans/enzimología , Ataxina-7/genética , Ceguera/genética , Enzimas Desubicuitinizantes/genética , Drosophila/enzimología , Histonas/metabolismo , Humanos , Ratones , Complejos Multienzimáticos/genética , Mutación , Neoplasias/genética , Enfermedades Neurodegenerativas/genética , Péptidos/genética , Procesamiento Proteico-Postraduccional , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/enzimología , Transactivadores/genética , Factores de Transcripción p300-CBP/genéticaRESUMEN
Atxn7, a subunit of SAGA chromatin remodeling complex, is subject to polyglutamine expansion at the amino terminus, causing spinocerebellar ataxia type 7 (SCA7), a progressive retinal and neurodegenerative disease. Within SAGA, the Atxn7 amino terminus anchors Non-stop, a deubiquitinase, to the complex. To understand the scope of Atxn7-dependent regulation of Non-stop, substrates of the deubiquitinase were sought. This revealed Non-stop, dissociated from Atxn7, interacts with Arp2/3 and WAVE regulatory complexes (WRC), which control actin cytoskeleton assembly. There, Non-stop countered polyubiquitination and proteasomal degradation of WRC subunit SCAR. Dependent on conserved WRC interacting receptor sequences (WIRS), Non-stop augmentation increased protein levels, and directed subcellular localization, of SCAR, decreasing cell area and number of protrusions. In vivo, heterozygous mutation of SCAR did not significantly rescue knockdown of Atxn7, but heterozygous mutation of Atxn7 rescued haploinsufficiency of SCAR.