Biophysics Reports

2017 Vol. 3, No. 4-6

Cover Story

Mitochondrion is the main intracellular site for thermogenesis and attractive energy expenditure targeting for obesity therapy.Here,the authors 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 norepinephrineinduced thermogenesis is low,and measure the maximum transient rate of temperature increase in brown adipocytes.

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Class C G protein-coupled receptors:reviving old couples with new partners

Thor C. Møller, David Moreno-Delgado, Jean-Philippe Pin, Julie Kniazeff

2017, 3(4-6): 57-63. doi: 10.1007/s41048-017-0036-9

Abstract(727)

PDF(141)

Abstract:
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.

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

2017, 3(4-6): 64-72. doi: 10.1007/s41048-017-0037-8

Abstract(685)

PDF(243)

Abstract:
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.

Energy coupling mechanisms of AcrB-like RND transporters

Xuejun C. Zhang, Min Liu, Lei Han

2017, 3(4-6): 73-84. doi: 10.1007/s41048-017-0042-y

Abstract(613)

PDF(274)

Abstract:
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

2017, 3(4-6): 85-91. doi: 10.1007/s41048-017-0039-6

Abstract(508)

PDF(282)

Abstract:
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.

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

2017, 3(4-6): 92-99. doi: 10.1007/s41048-017-0043-x

Abstract(178)

PDF(301)

Abstract:
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.

Protocol for analyzing protein ensemble structures from chemical cross-links using DynaXL

Zhou Gong, Zhu Liu, Xu Dong, Yue-He Ding, Meng-Qiu Dong, Chun Tang

2017, 3(4-6): 100-108. doi: 10.1007/s41048-017-0044-9

Abstract(146)

PDF(185)

Abstract:
Chemical cross-linking coupled with mass spectroscopy (CXMS) is a powerful technique for investigating protein structures. CXMS has been mostly used to characterize the predominant structure for a protein, whereas cross-links incompatible with a unique structure of a protein or a protein complex are often discarded. We have recently shown that the so-called over-length cross-links actually contain protein dynamics information. We have thus established a method called DynaXL, which allow us to extract the information from the over-length cross-links and to visualize protein ensemble structures. In this protocol, we present the detailed procedure for using DynaXL, which comprises five steps. They are identification of highly confident cross-links, delineation of protein domains/subunits, ensemble rigidbody refinement, and final validation/assessment. The DynaXL method is generally applicable for analyzing the ensemble structures of multi-domain proteins and protein-protein complexes, and is freely available at www.tanglab.org/resources.