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2.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361009

RESUMO

The parietal cortex of rodents participates in sensory and spatial processing, movement planning, and decision-making, but much less is known about its role in associative learning and memory formation. The present study aims to examine the involvement of the parietal association cortex (PtA) in associative fear memory acquisition and retrieval in mice. Using ex vivo c-Fos immunohistochemical mapping and in vivo Fos-EGFP two-photon imaging, we show that PtA neurons were specifically activated both during acquisition and retrieval of cued fear memory. Fos immunohistochemistry revealed specific activation of the PtA neurons during retrieval of the 1-day-old fear memory. In vivo two-photon Fos-EGFP imaging confirmed this result and in addition detected specific c-Fos responses of the PtA neurons during acquisition of cued fear memory. To allow a more detailed study of the long-term activity of such PtA engram neurons, we generated a Fos-Cre-GCaMP transgenic mouse line that employs the Targeted Recombination in Active Populations (TRAP) technique to detect calcium events specifically in cells that were Fos-active during conditioning. We show that gradual accumulation of GCaMP3 in the PtA neurons of Fos-Cre-GCaMP mice peaks at the 4th day after fear learning. We also describe calcium transients in the cell bodies and dendrites of the TRAPed neurons. This provides a proof-of-principle for TRAP-based calcium imaging of PtA functions during memory processes as well as in experimental models of fear- and anxiety-related psychiatric disorders and their specific therapies.


Assuntos
Medo , Memória , Lobo Parietal/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Aprendizagem por Associação , Sinalização do Cálcio , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Lobo Parietal/citologia , Lobo Parietal/fisiologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445257

RESUMO

The production of pancreatic ß cells is the most challenging step for curing diabetes using next-generation treatments. Adult pancreatic endocrine cells are thought to be maintained by the self-duplication of differentiated cells, and pancreatic endocrine neogenesis can only be observed when the tissue is severely damaged. Experimentally, this can be performed using a method named partial duct ligation (PDL). As the success rate of PDL surgery is low because of difficulties in identifying the pancreatic duct, we previously proposed a method for fluorescently labeling the duct in live animals. Using this method, we performed PDL on neurogenin3 (Ngn3)-GFP transgenic mice to determine the origin of endocrine precursor cells and evaluate their potential to differentiate into multiple cell types. Ngn3-activated cells, which were marked with GFP, appeared after PDL operation. Because some GFP-positive cells were aligned proximally to the duct, we hypothesized that Ngn3-positive cells arise from the pancreatic duct. Therefore, we next developed an in vitro pancreatic duct culture system using Ngn3-GFP mice and examined whether Ngn3-positive cells emerge from this duct. We observed GFP expressions in ductal organoid cultures. GFP expressions were correlated with Ngn3 expressions and endocrine cell lineage markers. Interestingly, tuft cell markers were also correlated with GFP expressions. Our results demonstrate that in adult mice, Ngn3-positive endocrine precursor cells arise from the pancreatic ducts both in vivo and in vitro experiments indicating that the pancreatic duct could be a potential donor for therapeutic use.


Assuntos
Antígenos de Diferenciação/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ductos Pancreáticos/metabolismo , Células-Tronco/metabolismo , Animais , Antígenos de Diferenciação/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Organoides/citologia , Organoides/metabolismo , Ductos Pancreáticos/citologia , Células-Tronco/citologia
4.
Anal Chem ; 93(35): 12011-12021, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34428029

RESUMO

Compartmentalization and integration of molecular processes through diffusion are basic mechanisms through which cells perform biological functions. To characterize these mechanisms in live cells, quantitative and ultrasensitive analytical methods with high spatial and temporal resolution are needed. Here, we present quantitative scanning-free confocal microscopy with single-molecule sensitivity, high temporal resolution (∼10 µs/frame), and fluorescence lifetime imaging capacity, developed by integrating massively parallel fluorescence correlation spectroscopy with fluorescence lifetime imaging microscopy (mpFCS/FLIM); we validate the method, use it to map in live cell location-specific variations in the concentration, diffusion, homodimerization, DNA binding, and local environment of the oligodendrocyte transcription factor 2 fused with the enhanced Green Fluorescent Protein (OLIG2-eGFP), and characterize the effects of an allosteric inhibitor of OLIG2 dimerization on these determinants of OLIG2 function. In particular, we show that cytoplasmic OLIG2-eGFP is largely monomeric and freely diffusing, with the fraction of freely diffusing OLIG2-eGFP molecules being fD,freecyt = (0.75 ± 0.10) and the diffusion time τD,freecyt = (0.5 ± 0.3) ms. In contrast, OLIG2-eGFP homodimers are abundant in the cell nucleus, constituting ∼25% of the nuclear pool, some fD,boundnuc = (0.65 ± 0.10) of nuclear OLIG2-eGFP is bound to chromatin DNA, whereas freely moving OLIG2-eGFP molecules diffuse at the same rate as those in the cytoplasm, as evident from the lateral diffusion times τD,freenuc = τD,freecyt = (0.5 ± 0.3) ms. OLIG2-eGFP interactions with chromatin DNA, revealed through their influence on the apparent diffusion behavior of OLIG2-eGFP, τD,boundnuc (850 ± 500) ms, are characterized by an apparent dissociation constant Kd,appOLIG2-DNA = (45 ± 30) nM. The apparent dissociation constant of OLIG2-eGFP homodimers was estimated to be Kd,app(OLIG2-eGFP)2 ≈ 560 nM. The allosteric inhibitor of OLIG2 dimerization, compound NSC 50467, neither affects OLIG2-eGFP properties in the cytoplasm nor does it alter the overall cytoplasmic environment. In contrast, it significantly impedes OLIG2-eGFP homodimerization in the cell nucleus, increasing five-fold the apparent dissociation constant, Kd,app,NSC50467(OLIG2-eGFP)2 ≈ 3 µM, thus reducing homodimer levels to below 7% and effectively abolishing OLIG2-eGFP specific binding to chromatin DNA. The mpFCS/FLIM methodology has a myriad of applications in biomedical research and pharmaceutical industry. For example, it is indispensable for understanding how biological functions emerge through the dynamic integration of location-specific molecular processes and invaluable for drug development, as it allows us to quantitatively characterize the interactions of drugs with drug targets in live cells.


Assuntos
Núcleo Celular , Proteínas de Fluorescência Verde/genética , Microscopia Confocal , Microscopia de Fluorescência , Fator de Transcrição 2 de Oligodendrócitos , Espectrometria de Fluorescência
5.
Bioengineered ; 12(1): 4407-4419, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34436976

RESUMO

Widespread infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has led to a global pandemic. Currently, various approaches are being taken up to develop vaccines and therapeutics to treat SARS-CoV2 infection. Consequently, the S protein has become an important target protein for developing vaccines and therapeutics against SARS-CoV2. However, the highly infective nature of SARS-CoV2 restricts experimentation with the virus to highly secure BSL3 facilities. The availability of fusion-enabled, nonreplicating, and nonbiohazardous mimics of SARS-CoV2 virus fusion, containing the viral S or S and M protein in their native conformation on mammalian cells, can serve as a useful substitute for studying viral fusion for testing various inhibitors of viral fusion. This would avoid the use of the BSL3 facility for fusion studies required to develop therapeutics. In the present study, we have developed SARS-CoV2 virus fusion mimics (SCFMs) using mammalian cells transfected with constructs coding for S or S and M protein. The fusogenic property of the mimic(s) and their interaction with the functional human ACE2 receptors was confirmed experimentally. We have also shown that such mimics can easily be used in an inhibition assay. These mimic(s) can be easily prepared on a large scale, and such SCFMs can serve as an invaluable resource for viral fusion inhibition assays and in vitro screening of antiviral agents, which can be shared/handled between labs/facilities without worrying about any biohazard while working under routine laboratory conditions, avoiding the use of BSL3 laboratory.Abbreviations :SCFM: SARS-CoV2 Virus Fusion Mimic; ACE2: Angiotensin-Converting Enzyme 2; hACE2: Human Angiotensin-Converting enzyme 2; MEF: Mouse Embryonic Fibroblasts; HBSS: Hanks Balanced Salt Solution; FBS: Fetal Bovine Serum.


Assuntos
Anticorpos Neutralizantes/farmacologia , Contenção de Riscos Biológicos/métodos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Proteínas da Matriz Viral/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Chlorocebus aethiops , Embrião de Mamíferos , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células MCF-7 , Camundongos , Mimetismo Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , Receptores Virais/genética , Receptores Virais/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção , Células Vero , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
6.
Am J Vet Res ; 82(9): 770-776, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34432512

RESUMO

OBJECTIVE: To characterize the ultrastructure of mesenchymal stem cells (MSCs) that were harvested from the adipose tissue (AT-MSCs) and bone marrow (BM-MSCs) of horses and transfected with green fluorescent protein. SAMPLE: MSCs from adipose tissue and bone marrow of 6 adult female Hispano-Bretón horses. PROCEDURES: Harvested equine MSCs were cultivated and transfected with green fluorescent protein, and the immunophenotypes of the MSCs were characterized by use of anti-CD90 and anti-CD105 monoclonal antibodies. When stable transfection of MSCs was achieved, the morphological and ultrastructural characteristics of transfected and nontransfected AT-MSCs and BM-MSCs were compared with electron microscopy. RESULTS: The protocols for transfection and subsequent isolation of transfected cells with use of G418 were suitable for obtaining transfected MSCs. Transfection efficiency was 5% in AT-MSCs and 4% in BM-MSCs. Characterization of transfected and nontransfected MSCs revealed that they share immunocytochemical and morphological profiles. Expression of CD90 was significantly higher for transfected versus nontransfected AT-MSCs (97% vs 92%). Expression of CD105 was significantly lower for transfected versus nontransfected BM-MSCs (85% vs 94%). Transfected BM-MSCs had differences in organelles, compared with the other cell types, specifically including most commonly the rough endoplasmic reticulum with dilated cisternae and mitochondria. CONCLUSION AND CLINICAL RELEVANCE: These findings contribute to the knowledge base of the characteristics of equine AT-MSCs and BM-MSCs and of transfected versus nontransfected equine MSCs. The data provided a valuable starting point for researchers wishing to further study the morphological characteristics of equine MSCs.


Assuntos
Células-Tronco Mesenquimais , Tecido Adiposo , Animais , Medula Óssea , Células da Medula Óssea , Feminino , Proteínas de Fluorescência Verde/genética , Cavalos
7.
Methods Mol Biol ; 2312: 237-251, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228294

RESUMO

Chemical control of protein localization is a powerful approach for manipulating mammalian cellular processes. Self-localizing ligand-induced protein translocation (SLIPT) is an emerging platform that enables control of protein localization in living mammalian cells using synthetic self-localizing ligands (SLs). We recently established a chemogenetic SLIPT system, in which any protein of interest fused to an engineered variant of Escherichia coli dihydrofolate reductase, DHFRiK6, can be rapidly and specifically translocated from the cytoplasm to the inner leaflet of the plasma membrane (PM) using a trimethoprim (TMP)-based PM-targeting SL, mDcTMP. The mDcTMP-mediated PM recruitment of DHFRiK6-fusion proteins can be efficiently returned to the cytoplasm by subsequent addition of free TMP, enabling temporal and reversible control over the protein localization. Here we describe the use of this mDcTMP/DHFRiK6-based SLIPT system for inducing (1) reversible protein translocation and (2) synthetic activation of the Raf/ERK pathway. This system provides a simple and versatile tool in mammalian synthetic biology for temporally manipulating various signaling molecules and pathways at the PM.


Assuntos
Engenharia Celular , Proteínas de Escherichia coli/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas Genéticas , Biologia Sintética , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/farmacologia , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tetra-Hidrofolato Desidrogenase/metabolismo , Quinases raf/metabolismo
8.
Methods Mol Biol ; 2312: 277-285, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228296

RESUMO

There are increasing evidence and growing interest in the relationship between protein aggregates/phase separation and various human diseases, especially neurodegenerative diseases. However, we do not entirely comprehend how aggregates generate or the clearance network of chaperones, proteasomes, ubiquitin ligases, and other factors interact with aggregates. Here, we describe chemically controllable systems compose with a genetically engineered cell and a small drug that enables us to rapidly induce protein aggregates' formation by withdrawing the small molecule. This trigger does not activate global stress responses induced by stimuli, such as proteasome inhibitors or heat shock. This method can produce aggregates in a specific compartment and diverse experimental systems, including live animals.


Assuntos
Engenharia Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Engenharia de Proteínas , Proteínas de Ligação a Tacrolimo/genética , Animais , Técnicas de Cultura de Células , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência , Mutação , Células NIH 3T3 , Agregados Proteicos , Estabilidade Proteica , Proteostase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Transfecção
9.
Methods Mol Biol ; 2312: 309-320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228299

RESUMO

Developments in genome-editing technology, especially CRISPR-Cas9, have revolutionized the way in which genetically engineered animals are generated. However, the process of generation includes microinjection to the one-cell stage embryo and the transfer of the microinjected embryo to the surrogate animals, which requires trained personnel. We recently reported the method includes introduction of CRISPR-Cas9 systems to the developing cerebral cortex via in utero electroporation thus generating gene-targeted neural stem cells in vivo. This technique is widely applicable for gene knockout, monitoring gene expression, and lineage analysis in developmental biology. In this chapter, the detailed protocol of EGFP (enhanced green fluorescent protein) knock-in method via in utero electroporation is described.


Assuntos
Encéfalo/metabolismo , Eletroporação , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Técnicas de Transferência de Genes , Animais , Encéfalo/embriologia , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Técnicas de Introdução de Genes , Genes Reporter , Idade Gestacional , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Gravidez , RNA Guia/genética , RNA Guia/metabolismo
10.
Nucleic Acids Res ; 49(13): 7775-7790, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197613

RESUMO

CRISPR Cas12a is an RNA-programmable endonuclease particularly suitable for gene regulation. This is due to its preference for T-rich PAMs that allows it to more easily target AT-rich promoter sequences, and built-in RNase activity which can process a single CRISPR RNA array encoding multiple spacers into individual guide RNAs (gRNAs), thereby simplifying multiplexed gene regulation. Here, we develop a flexible dCas12a-based CRISPRi system for Saccharomyces cerevisiae and systematically evaluate its design features. This includes the role of the NLS position, use of repression domains, and the position of the gRNA target. Our optimal system is comprised of dCas12a E925A with a single C-terminal NLS and a Mxi1 or a MIG1 repression domain, which enables up to 97% downregulation of a reporter gene. We also extend this system to allow for inducible regulation via an RNAP II-controlled promoter, demonstrate position-dependent effects in crRNA arrays, and use multiplexed regulation to stringently control a heterologous ß-carotene pathway. Together these findings offer valuable insights into the design constraints of dCas12a-based CRISPRi and enable new avenues for flexible and efficient gene regulation in S. cerevisiae.


Assuntos
Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Endodesoxirribonucleases/química , Regulação da Expressão Gênica , Saccharomyces cerevisiae/genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Regulação para Baixo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Fluorescência Verde/genética , Sinais de Localização Nuclear , Regiões Promotoras Genéticas , Domínios Proteicos , RNA/metabolismo , RNA Polimerase II/metabolismo , beta Caroteno/biossíntese
11.
Nat Cell Biol ; 23(8): 881-893, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34326481

RESUMO

The 11 zinc finger (ZF) protein CTCF regulates topologically associating domain formation and transcription through selective binding to thousands of genomic sites. Here, we replaced endogenous CTCF in mouse embryonic stem cells with green-fluorescent-protein-tagged wild-type or mutant proteins lacking individual ZFs to identify additional determinants of CTCF positioning and function. While ZF1 and ZF8-ZF11 are not essential for cell survival, ZF8 deletion strikingly increases the DNA binding off-rate of mutant CTCF, resulting in reduced CTCF chromatin residence time. Loss of ZF8 results in widespread weakening of topologically associating domains, aberrant gene expression and increased genome-wide DNA methylation. Thus, important chromatin-templated processes rely on accurate CTCF chromatin residence time, which we propose depends on local sequence and chromatin context as well as global CTCF protein concentration.


Assuntos
Fator de Ligação a CCCTC/fisiologia , Cromatina/metabolismo , Metilação de DNA , Regulação da Expressão Gênica , Genoma , Células-Tronco Pluripotentes/fisiologia , Animais , Fator de Ligação a CCCTC/genética , Feminino , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Mitose , Células-Tronco Embrionárias Murinas , Mutação , Células-Tronco Pluripotentes/metabolismo , Fatores de Tempo , Elongação da Transcrição Genética
12.
Commun Biol ; 4(1): 841, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230602

RESUMO

Characterizing protein-protein interactions (PPIs) is an effective method to help explore protein function. Here, through integrating a newly identified split human Rhinovirus 3 C (HRV 3 C) protease, super-folder GFP (sfGFP), and ClpXP-SsrA protein degradation machinery, we developed a fluorescence-assisted single-cell methodology (split protease-E. coli ClpXP (SPEC)) to explore protein-protein interactions for both eukaryotic and prokaryotic species in E. coli cells. We firstly identified a highly efficient split HRV 3 C protease with high re-assembly ability and then incorporated it into the SPEC method. The SPEC method could convert the cellular protein-protein interaction to quantitative fluorescence signals through a split HRV 3 C protease-mediated proteolytic reaction with high efficiency and broad temperature adaptability. Using SPEC method, we explored the interactions among effectors of representative type I-E and I-F CRISPR/Cas complexes, which combining with subsequent studies of Cas3 mutations conferred further understanding of the functions and structures of CRISPR/Cas complexes.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mapas de Interação de Proteínas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Endopeptidase Clp/genética , Enterovirus/enzimologia , Enterovirus/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteólise , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
13.
Nat Commun ; 12(1): 4504, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301936

RESUMO

Genes are expressed in stochastic transcriptional bursts linked to alternating active and inactive promoter states. A major challenge in transcription is understanding how promoter composition dictates bursting, particularly in multicellular organisms. We investigate two key Drosophila developmental promoter motifs, the TATA box (TATA) and the Initiator (INR). Using live imaging in Drosophila embryos and new computational methods, we demonstrate that bursting occurs on multiple timescales ranging from seconds to minutes. TATA-containing promoters and INR-containing promoters exhibit distinct dynamics, with one or two separate rate-limiting steps respectively. A TATA box is associated with long active states, high rates of polymerase initiation, and short-lived, infrequent inactive states. In contrast, the INR motif leads to two inactive states, one of which relates to promoter-proximal polymerase pausing. Surprisingly, the model suggests pausing is not obligatory, but occurs stochastically for a subset of polymerases. Overall, our results provide a rationale for promoter switching during zygotic genome activation.


Assuntos
Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Regiões Promotoras Genéticas/genética , TATA Box/genética , Imagem com Lapso de Tempo/métodos , Transcrição Genética/genética , Algoritmos , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/embriologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Modelos Teóricos
14.
Nat Commun ; 12(1): 4511, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301939

RESUMO

Septins are cytoskeletal proteins that assemble into hetero-oligomeric complexes and sense micron-scale membrane curvature. During infection with Shigella flexneri, an invasive enteropathogen, septins restrict actin tail formation by entrapping bacteria in cage-like structures. Here, we reconstitute septin cages in vitro using purified recombinant septin complexes (SEPT2-SEPT6-SEPT7), and study how these recognize bacterial cells and assemble on their surface. We show that septin complexes recognize the pole of growing Shigella cells. An amphipathic helix domain in human SEPT6 enables septins to sense positively curved membranes and entrap bacterial cells. Shigella strains lacking lipopolysaccharide components are more efficiently entrapped in septin cages. Finally, cryo-electron tomography of in vitro cages reveals how septins assemble as filaments on the bacterial cell surface.


Assuntos
Actinas/metabolismo , Disenteria Bacilar/metabolismo , Proteínas Recombinantes/metabolismo , Septinas/metabolismo , Shigella flexneri/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Disenteria Bacilar/microbiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lipopolissacarídeos/metabolismo , Microscopia de Fluorescência , Mutação , Ligação Proteica , Septinas/genética , Shigella flexneri/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Molecules ; 26(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203222

RESUMO

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.


Assuntos
Proteínas de Bactérias/química , Proteínas de Fluorescência Verde/química , Corpos de Inclusão/química , Fosfolipases A1/química , Proteínas Recombinantes de Fusão/química , Yersinia pseudotuberculosis/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Fosfolipases A1/biossíntese , Fosfolipases A1/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Yersinia pseudotuberculosis/enzimologia
16.
Nat Commun ; 12(1): 4327, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267202

RESUMO

Trivalent rare earth elements (REEs) are widely used in agriculture. Aerially applied REEs enter leaf epidermal cells by endocytosis and act systemically to improve the growth of the whole plant. The mechanistic basis of their systemic activity is unclear. Here, we show that treatment of Arabidopsis leaves with trivalent lanthanum [La(III)], a representative of REEs, triggers systemic endocytosis from leaves to roots. La(III)-induced systemic endocytosis requires AtrbohD-mediated reactive oxygen species production and jasmonic acid. Systemic endocytosis impacts the accumulation of mineral elements and the development of roots consistent with the growth promoting effects induced by aerially applied REEs. These findings provide insights into the mechanistic basis of REE activity in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Lantânio/farmacologia , NADPH Oxidases/metabolismo , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Ciclopentanos/metabolismo , Endocitose/fisiologia , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Minerais/metabolismo , NADPH Oxidases/genética , Oxilipinas/metabolismo , Células Vegetais/efeitos dos fármacos , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Transdução de Sinais
17.
Appl Microbiol Biotechnol ; 105(13): 5529-5539, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34254155

RESUMO

Aspergillus niger is widely used for the efficient production of organic acids and enzyme preparations. However, this organism lacks basic genetic elements for dynamic control, especially inducible promoters that can respond to specific environmental signals. Since these are desirable for better adaptation of fermentation to large-scale industrial production, herein, we have identified the two first hypoxia-inducible promoters in A. niger, PsrbB and PfhbA. Their performance under high or low oxygen conditions was monitored using two reporter proteins, green fluorescent protein (EGFP) and ß-glucuronidase (GUS). For comparison, basal expression of the general strong promoter PgpdA was lower than PsrbB but higher than PfhbA. However, under hypoxia, both promoters showed higher expression than under hyperoxia, and these values were also higher than those observed for PgpdA. For PsrbB, strength under hypoxia was ~2-3 times higher than under hyperoxia (for PfhbA, 3-9 times higher) and ~2.5-5 times higher than for PgpdA (for PfhbA, 2-3 times higher). Promoter truncation analysis showed that the PsrbB fragment -1024 to -588 bp is the core region that determines hypoxia response. KEY POINTS: The first identification of two hypoxia-inducible promoters in A. niger is a promising tool for modulation of target genes under hypoxia. Two reporter genes revealed a different activity and responsiveness to hypoxia of PfhbA and PsrbB promoters, which is relevant for the development of dynamic metabolic regulation of A. niger fermentation. PsrbB promoter truncation and bioinformatics analysis is the foundation for further research.


Assuntos
Aspergillus niger , Hipóxia , Aspergillus niger/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Humanos , Regiões Promotoras Genéticas
18.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201604

RESUMO

The spotted-wing Drosophila (Drosophila suzukii Matsumura) is native to eastern Asia, but has become a global threat to fruit production. In recent years, CRISPR/Cas9 targeting was established in this species allowing for functional genomic and genetic control studies. Here, we report the generation and characterization of Cas9-expressing strains of D. suzukii. Five independent transgenic lines were generated using a piggyBac construct containing the EGFP fluorescent marker gene and the Cas9 gene under the control of the D. melanogaster heat shock protein 70 promoter and 3'UTR. Heat-shock (HS) treated embryos were analyzed by reverse transcriptase PCR, revealing strong heat inducibility of the transgenic Cas9 expression. By injecting gRNA targeting EGFP into one selected line, 50.0% of G0 flies showed mosaic loss-of-fluorescence phenotype, and 45.5% of G0 flies produced G1 mutants without HS. Such somatic and germline mutagenesis rates were increased to 95.4% and 85.7%, respectively, by applying a HS. Parental flies receiving HS resulted in high inheritance of the mutation (92%) in their progeny. Additionally, targeting the endogenous gene yellow led to the lack of pigmentation and male lethality. We discuss the potential use of these efficient and temperature-dependent Cas9-expressing strains for the genetic studies in D. suzukii.


Assuntos
Sistemas CRISPR-Cas , Drosophila/genética , Marcação de Genes/métodos , Animais , Animais Geneticamente Modificados , Proteína 9 Associada à CRISPR/genética , Drosophila/embriologia , Proteínas de Drosophila/genética , Embrião não Mamífero , Feminino , Proteínas de Fluorescência Verde/genética , Resposta ao Choque Térmico/genética , Masculino , Mutagênese , Pigmentação/genética , Temperatura , Transgenes
19.
Food Chem ; 361: 129829, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34087571

RESUMO

To detect major soy isoflavone glycosides, namely daidzin (DZ) and genistin (GEN), novel open sandwich fluorescence-linked immunosorbent assay (os-FLISA) was developed by taking advantage of enhanced interactions between variable regions of heavy (VH) and light chain (VL) domains in the presence of an antigen. The VH and VL genes were expressed in Escherichia coli as a chimera protein with green fluorescence protein (AcGFP1) and maltose-binding protein (MBP), respectively. Comprehensive characterization of os-FLISA displayed nearly the same specificity as parental DZ- and GEN-specific monoclonal antibody, demonstrating the potential of the developed assay for detection of both DZ and GEN. Their detectable range in this system exhibited at 0.1-12.5 µg mL-1. Subsequent validation analysis revealed that os-FLISA was reliable and accurate system for detection of total soy isoflavone glycosides. Notably, this is the first FLISA based on an open sandwich system, which can be employed for the detection of small molecules.


Assuntos
Técnicas de Imunoadsorção , Isoflavonas/análise , Soja/química , Anticorpos Monoclonais/genética , Escherichia coli/genética , Fluorescência , Análise de Alimentos/métodos , Proteínas de Fluorescência Verde/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Isoflavonas/imunologia , Limite de Detecção , Proteínas Ligantes de Maltose/genética , Proteínas Recombinantes/genética , Reprodutibilidade dos Testes
20.
Methods Mol Biol ; 2310: 113-159, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34096002

RESUMO

Mitochondria are dynamic organelles that participate in a broad array of molecular functions within the cell. They are responsible for maintaining the appropriate energetic levels and control the cellular homeostasis throughout the generation of intermediary metabolites. Preserving a healthy and functional mitochondrial population is of fundamental importance throughout the life of the cells under pathophysiological conditions. Hence, cells have evolved fine-tuned mechanisms of quality control that help to preserve the right amount of functional mitochondria to meet the demand of the cell. The specific recycling of mitochondria by autophagy, termed mitophagy, represents the primary contributor to mitochondrial quality control. During this process, damaged or unnecessary mitochondria are recognized and selectively degraded. In the past few years, the knowledge in mitophagy has seen rapid progress, and a growing body of evidence confirms that mitophagy holds a central role in controlling cellular functions and the progression of various human diseases.In this chapter, we will discuss the pathophysiological roles of mitophagy and provide a general overview of the current methods used to monitor and quantify mitophagy. We will also outline the main established approaches to investigate the mitochondrial function, metabolism, morphology, and protein damage.


Assuntos
Doenças Cardiovasculares/patologia , Microscopia Confocal , Microscopia de Fluorescência , Mitocôndrias/patologia , Dinâmica Mitocondrial , Mitofagia , Neoplasias/patologia , Doenças Neurodegenerativas/patologia , Animais , Biomarcadores/metabolismo , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Linhagem Celular , Metabolismo Energético , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Transfecção
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