RESUMO
Single-molecule fluorescence and in particular single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful tool to provide real-time information on the dynamic architecture of large macromolecular structures such as eukaryotic transcription initiation complexes. In contrast to other structural biology methods, not only structural details, but dynamics transitions are revealed thus closing in on the underlying molecular mechanisms. Here, we describe a comprehensive quantitative biophysical toolbox which can be used for rigorous analysis of dynamic protein-nucleic acid complexes and is applied to the study of eukaryotic transcription initiation. We present detailed protocols for the purification of all essential protein components of the minimal eukaryotic transcription initiation complex. Moreover, we demonstrate how elaborate strategies for site-specific protein labeling can be used to produce complexes with dye molecules attached to arbitrary desired positions. These complexes are then used for smFRET measurements. Moreover, we describe the Nano-Positioning System (NPS) which allows us to quantitatively use the results from a network of smFRET measurements to obtain structural information. With this we provide a toolbox to answer open questions which could not be addressed using methods like X-ray crystallography or cryo-electron microscopy.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Complexos Multiproteicos/química , RNA Polimerase II/química , Imagem Individual de Molécula/métodos , Iniciação da Transcrição Genética , Algoritmos , Microscopia Crioeletrônica , Transferência Ressonante de Energia de Fluorescência/instrumentação , Cinética , Microscopia de Fluorescência/instrumentação , Complexos Multiproteicos/metabolismo , Peptídeos/química , Regiões Promotoras Genéticas , Estrutura Quaternária de Proteína , RNA Polimerase II/genética , RNA Polimerase II/isolamento & purificação , RNA Polimerase II/metabolismo , Imagem Individual de Molécula/instrumentação , Coloração e Rotulagem/instrumentação , Coloração e Rotulagem/métodosRESUMO
During osteoporosis bone formation by osteoblasts is reduced and/or bone resorption by osteoclasts is enhanced. Currently, only a few factors have been identified in the regulation of bone integrity by osteoblast-derived osteocytes. In this study, we show that specific disruption of menin, encoded by multiple endocrine neoplasia type 1 (Men1), in osteoblasts and osteocytes caused osteoporosis despite the preservation of osteoblast differentiation and the bone formation rate. Instead, an increase in osteoclast numbers and bone resorption was detected that persisted even when the deletion of Men1 was restricted to osteocytes. We demonstrate that isolated Men1-deficient osteocytes expressed numerous soluble mediators, such as C-X-C motif chemokine 10 (CXCL10), and that CXCL10-mediated osteoclastogenesis was reduced by CXCL10-neutralizing antibodies. Collectively, our data reveal a novel role for Men1 in osteocyte-osteoclast crosstalk by controlling osteoclastogenesis through the action of soluble factors. A role for Men1 in maintaining bone integrity and thereby preventing osteoporosis is proposed.
