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To be launched on Aug 2015
Biophysical Society of China
Institute of Biophysics, CAS, P.R. China.
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Determining the target protein localization in 3D using the combination of FIB-SEM and APEX2
Yang Shi, Li Wang, Jianguo Zhang, Yujia Zhai, Fei Sun
Biophysics Reports DOI: 10.1007/s41048-017-0043-x
Determining the cellular localization of proteins of interest at nanometer resolution is necessary for elucidating their functions. Besides super-resolution fluorescence microscopy, conventional electron microscopy (EM) combined with immunolabeling or clonable EM tags provides a unique approach to correlate protein localization information and cellular ultrastructural information. However, there are still rare cases of such correlation in three-dimensional (3D) spaces. Here, we developed an approach by combining the focus ion beam scanning electron microscopy (FIB-SEM) and a promising clonable EM tag APEX2(an enhanced ascorbate peroxidase 2) to determine the target protein localization within 3D cellular ultrastructural context. We further utilized this approach to study the 3D localization of mitochondrial dynamics-related proteins (MiD49/51, Mff, Fis1, and Mfn2) in the cells where the target proteins were overexpressed. We found that all the target proteins were located at the surface of the mitochondrial outer membrane accompanying with mitochondrial clusters. Mid49/51, Mff, and hFis1 spread widely around the mitochondrial surface while Mfn2 only exists at the contact sites.
Energy coupling mechanisms of AcrB-like RND transporters
Xuejun C. Zhang, Min Liu, Lei Han
Biophysics Reports DOI: 10.1007/s41048-017-0042-y
Prokaryotic AcrB-like proteins belong to a family of transporters of the RND superfamily, and as main contributing factor to multidrug resistance pose a tremendous threat to future human health. A unique feature of AcrB transporters is the presence of two separate domains responsible for carrying substrate and generating energy. Significant progress has been made in elucidating the three-dimensional structures of the homo-trimer complexes of AcrB-like transporters, and a three-step functional rotation was identified for this class of transporters. However, the detailed mechanisms for the transduction of the substrate binding signal, as well as the energy coupling processes between the functionally distinct domains remain to be established. Here, we propose a model for the interdomain communication in AcrB that explains how the substrate binding signal from the substrate-carrier domain triggers protonation in the transmembrane domain. Our model further provides a plausible mechanism that explains how protonation induces conformational changes in the substrate-carrier domain. We summarize the thermodynamic principles that govern the functional cycle of the AcrB trimer complex.
Dye-based mito-thermometry and its application in thermogenesis of brown adipocytes
Tao-Rong Xie, Chun-Feng Liu, Jian-Sheng Kang
Biophysics Reports DOI: 10.1007/s41048-017-0039-6
Mitochondrion is the main intracellular site for thermogenesis and attractive energy expenditure targeting for obesity therapy. Here, we develop a method of mitochondrial thermometry based on Rhodamine B methyl ester, which equilibrates as a thermosensitive mixture of nonfluorescent and fluorescent resonance forms. Using this approach, we are able to demonstrate that the efficacy of norepinephrine-induced thermogenesis is low, and measure the maximum transient rate of temperature increase in brown adipocytes.
Choosing proper fluorescent dyes, proteins, and imaging techniques to study mitochondrial dynamics in mammalian cells
Xingguo Liu, Liang Yang, Qi Long, David Weaver, György Hajnóczky
Biophysics Reports DOI: 10.1007/s41048-017-0037-8
Mitochondrial dynamics refers to the processes maintaining mitochondrial homeostasis, including mitochondrial fission, fusion, transport, biogenesis, and mitophagy. Mitochondrial dynamics is essential for maintaining the metabolic function of mitochondria as well as their regulatory roles in cell signaling. In this review, we summarize the recently developed imaging techniques for studying mitochondrial dynamics including:mitochondrial-targeted fluorescent proteins and dyes, live-cell imaging using photoactivation, photoswitching and cell fusion, mitochondrial transcription and replication imaging by in situ hybridization, and imaging mitochondrial dynamics by super-resolution microscopy. Moreover, we discuss examples of how to choose and combine proper fluorescent dyes and/or proteins.
Class C G protein-coupled receptors: reviving old couples with new partners
Thor C. Møller, David Moreno-Delgado, Jean-Philippe Pin, Julie Kniazeff
Biophysics Reports DOI: 10.1007/s41048-017-0036-9
G protein-coupled receptors (GPCRs) are key players in cell communication and are encoded by the largest family in our genome. As such, GPCRs represent the main targets in drug development programs. Sequence analysis revealed several classes of GPCRs:the class A rhodopsin-like receptors represent the majority, the class B includes the secretin-like and adhesion GPCRs, the class F includes the frizzled receptors, and the class C includes receptors for the main neurotransmitters, glutamate and GABA, and those for sweet and umami taste and calcium receptors. Class C receptors are far more complex than other GPCRs, being mandatory dimers, with each subunit being composed of several domains. In this review, we summarize our actual knowledge regarding the activation mechanism and subunit organization of class C GPCRs, and how this brings information for many other GPCRs.
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