Biophysics Reports

2019 Vol. 5, No. 3

Cover Story

Cryo-electron tomography is an emerging electron microscopy technique for determining three-dimensional structures of cellular architectures near their native state at nanometer resolution, with a shortcoming of lack of specific labels. Fluorescence light microscopy, on the other hand, specifically visualizes target cellular and molecular components with fluorescent labels, but is limited to a reso-lution of 10 to 100 of nanometers. Combining the advantages of the two techniques, the authors have developed a cryocorrelative light and electron microscopy system. This system consists a custom-designed cryo-chamber that allows for fluorescence imaging of frozen-hydrated samples, and an algorithm to achieve accurate correlation. With this system and the optimized protocol, high-quality tomograms of neuronal synapses labelled by specific fluorescent tags in cultured hippocampal neurons are obtained at high efficiency.

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An efficient protocol of cryo-correlative light and electron microscopy for the study of neuronal synapses

Rong Sun, Yun-Tao Liu, Chang-Lu Tao, Lei Qi, Pak-Ming Lau, Z. Hong Zhou, Guo-Qiang Bi

2019, 5(3): 111-122. doi: 10.1007/s41048-019-0092-4

Abstract(164)

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Abstract:
Cryo-electron tomography is an emerging electron microscopy technique for determining threedimensional structures of cellular architectures near their native state at nanometer resolution, with a shortcoming of lack of specific labels. Fluorescence light microscopy, on the other hand, specifically visualizes target cellular and molecular components with fluorescent labels, but is limited to a resolution of tens to hundreds nanometers. Combining the advantages of the two techniques, we have developed a cryocorrelative light and electron microscopy system. Our system consists a custom-designed cryo-chamber that allows for fluorescence imaging of frozen-hydrated samples, and an algorithm to achieve accurate correlation. With this system and our optimized protocol, high-quality tomograms of neuronal synapses labelled by specific fluorescent tags in cultured hippocampal neurons are obtained at high efficiency.

Modeling the relationship of diverse genomic signatures to gene expression levels with the regulation of long-range enhancer-promoter interactions

Zhen-Xing Feng, Qian-Zhong Li, Jian-Jun Meng

2019, 5(3): 123-132. doi: 10.1007/s41048-019-0089-z

Abstract(105)

PDF(83)

Abstract:
Enhancer-promoter (E-P) interaction is an essential component of cis-regulatory regulation for gene expression. However, to comprehensively study the gene expression with the regulation of long-range E-P interactions is a major challenge in the regulatory networks. As these types of gene expression are regulated by diverse genomic signatures, we presented a computational method to study the relationships between gene expression levels and diverse genomic signatures. In this paper, based on the datasets of long-range E-P interactions, we extracted feature parameters from multiple signatures (e.g., epigenetic marks, transcription factors) and used regression models to predict the gene expression levels. In our results, we found that the predicted expression values correlated well with the measured expression values in both the interacting and non-interacting sets, and the correlation values of the interacting set were higher than that of the corresponding non-interacting set in each cell line, which indicated that the distal enhancers would cooperate with diverse genomic signatures to facilitate the expression level of target genes. By comparing the important signature features for the gene expression levels between the interacting and non-interacting sets in the same cell line, we found that the important specific signatures affect the gene expression regulated by distal enhancers. Our research provided additional insights about the roles of diverse signatures in gene expression with the regulation of distal enhancers.

Impact of bacterial chaperonin GroEL-GroES on bacteriorhodopsin folding and membrane integration

Xinwei Lu, Baomei Xu, Haiyan Sun, Junting Wei, Haixia Chi, Naseer Ullah Khan, Xiaojuan Wang, Xiaoqiang Wang, Fang Huang

2019, 5(3): 133-144. doi: 10.1007/s41048-019-0090-6

Abstract(86)

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Abstract:
Our understanding of molecular chaperone function in membrane protein biogenesis lags far behind that in soluble protein biogenesis. Through a combined approach including isothermal titration calorimetry, UV-Vis spectroscopy, and fluorescence spectroscopy, the behavior of ATP-dependent chaperonin GroEL-GroES, a paradigmatic chaperone of soluble protein folding, was investigated in the refolding of membrane protein bacteriorhodopsin (BR) and its membrane insertion. We found that BR bound asymmetrically to the double-ring GroEL, with a much higher affinity when it was partially denatured. GroEL alone showed a clear influence on BR refolding, but the presence of ATP was necessary to significantly enhance both the rate and yield of the GroEL-mediated folding, in contrast to the adverse effect of GroES on the folding yield. However, synergy between ATP and GroES was shown to be required not only for releasing high-affinity BR species from GroEL, but also for unfolding and rescuing the misfolded conformers complexed to GroEL. This is consistent with the observation that maximum rate enhancement of BR refolding or assembly with the prepared inverted membrane vesicles was achieved when the complete chaperonin system was used. Our results support the iterative unfolding mechanism of GroEL activity previously proposed for soluble proteins, whereby GroEL might perform repeated unfolding and release of BR, thus offering additional opportunities for timely folding or membrane integration. This work provides important information on the convergence of folding of membrane and soluble proteins in light of folding pathways and the role of molecular chaperones.

Perilipin 2 and lipid droplets provide reciprocal stabilization

Shimeng Xu, Fei Zou, Zhiqing Diao, Shuyan Zhang, Yaqin Deng, Xiaotong Zhu, Liujuan Cui, Jinhai Yu, Zhiguang Zhang, Adekunle Toyin Bamigbade, Hongchao Zhang, Xuan Wei, Xuelin Zhang, Bin Liang, Pingsheng Liu

2019, 5(3): 145-160. doi: 10.1007/s41048-019-0091-5

Abstract(399)

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Abstract:
The lipid droplet (LD)-associated protein adipose differentiation-related protein (ADRP or PLIN2) is required for the formation and stability of the LD organelle, whereas its biological roles are still obscure. Herein, we show that PLIN2 is the most abundant protein on the lipid droplets (LDs) of mouse myoblast cell line C2C12. Both the expression of PLIN2 and the accumulation of LDs were up-regulated in a time-and dose-dependent manner when the cells were treated with oleate (OA). The protein level of PLIN2 was positively correlated with the formation of LDs, suggesting that LDs stabilize PLIN2. Furthermore, knocking out PLIN2 in C2C12 cells led to enlarged LDs and higher triacylglycerol hydrolysis activity. The isolated PLIN2 null LDs became closely contact with mitochondria and other cellular organelles. Additionally, mitochondrial activity was suppressed by OA in PLIN2 null cells. Our results reveal the pivotal roles of PLIN2 in governing LD dynamics and their relationship to mitochondria, and suggest a reciprocal stabilization between PLIN2 and LDs.

Two chemical-controlled switchable Cas9s for tunable gene editing