RESUMO
Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Anticorpos de Domínio Único , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Citocinese , Corantes Fluorescentes/metabolismo , Mamíferos/metabolismo , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Anticorpos de Domínio Único/metabolismo , Tropomiosina/genética , Tropomiosina/metabolismoRESUMO
Nucleotide excision repair (NER) is the primary mechanism for removal of ultraviolet light (UV)-induced DNA photoproducts and is mechanistically conserved across all kingdoms of life. Bacterial NER involves damage recognition by UvrA2 and UvrB, followed by UvrC-mediated incision either side of the lesion. Here, using a combination of in vitro and in vivo single-molecule studies we show that a UvrBC complex is capable of lesion identification in the absence of UvrA. Single-molecule analysis of eGFP-labelled UvrB and UvrC in living cells showed that UV damage caused these proteins to switch from cytoplasmic diffusion to stable complexes on DNA. Surprisingly, ectopic expression of UvrC in a uvrA deleted strain increased UV survival. These data provide evidence for a previously unrealized mechanism of survival that can occur through direct lesion recognition by a UvrBC complex.
Assuntos
Adenosina Trifosfatases/genética , DNA Helicases/genética , Reparo do DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos da radiação , Adenosina Trifosfatases/deficiência , Bacillus/química , Bacillus/genética , Bacillus/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/deficiência , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Viabilidade Microbiana/genética , Viabilidade Microbiana/efeitos da radiação , Ligação Proteica , Imagem Individual de Molécula/métodos , Raios UltravioletaRESUMO
Single-molecule approaches to solving biophysical problems are powerful tools that allow static and dynamic real-time observations of specific molecular interactions of interest in the absence of ensemble-averaging effects. Here, we provide detailed protocols for building an experimental system that employs atomic force microscopy and a single-molecule DNA tightrope assay based on oblique angle illumination fluorescence microscopy. Together with approaches for engineering site-specific lesions into DNA substrates, these complementary biophysical techniques are well suited for investigating protein-DNA interactions that involve target-specific DNA-binding proteins, such as those engaged in a variety of DNA repair pathways. In this chapter, we demonstrate the utility of the platform by applying these techniques in the studies of proteins participating in nucleotide excision repair.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Microscopia de Força Atômica/métodos , Microscopia de Fluorescência/métodos , Animais , Sequência de Bases , DNA/análise , Proteínas de Ligação a DNA/análise , Desenho de Equipamento , Humanos , Microscopia de Força Atômica/instrumentação , Microscopia de Fluorescência/instrumentação , Ligação ProteicaRESUMO
Many protein interactions with DNA require specific sequences; however, how these sequences are located remains uncertain. DNA normally appears bundled in solution but, to study DNA-protein interactions, the DNA needs to be elongated. Using fluidics single DNA strands can be efficiently and rapidly elongated between beads immobilized on a microscope slide surface. Such "DNA tightropes" offer a valuable method to study protein search mechanisms. Real-time fluorescence imaging of these interactions provides quantitative descriptions of search mechanism at the single molecule level. In our lab, we use this method to study the complex process of nucleotide excision DNA repair to determine mechanisms of damage detection, lesion removal, and DNA excision.