ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Deubiquitinating Enzymes/metabolism , Multienzyme Complexes/metabolism , Trans-Activators/metabolism , Transcriptional Activation , p300-CBP Transcription Factors/metabolism , Animals , Arabidopsis/enzymology , Aspergillus nidulans/enzymology , Ataxin-7/genetics , Blindness/genetics , Deubiquitinating Enzymes/genetics , Drosophila/enzymology , Histones/metabolism , Humans , Mice , Multienzyme Complexes/genetics , Mutation , Neoplasms/genetics , Neurodegenerative Diseases/genetics , Peptides/genetics , Protein Processing, Post-Translational , RNA Polymerase II/metabolism , Saccharomyces cerevisiae/enzymology , Trans-Activators/genetics , p300-CBP Transcription Factors/geneticsABSTRACT
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.
