光激活荧光蛋白及其在植物分子细胞生物学研究中的应用

光激活荧光蛋白是指用特定光照射时,其荧光特性发生显著改变的一类荧光蛋白。借助光激活荧光蛋白的这种特性,可以实现对活细胞、细胞器或胞内分子的时空标记和追踪。该文介绍了目前光激活荧光蛋白的性质,并从多个方面对其应用进行了概括,包括分子标记与动态分析、蛋白质相互作用、细胞器及细胞组分动态研究、细胞追踪以及在光激活定位显微镜中的应用等,且对目前光激活荧光蛋白在植物分子细胞生物学中的应用进行了详细介绍。     

光激活荧光蛋白及其在植物分子细胞生物学研究中的应用

光激活荧光蛋白是指用特定光照射时, 其荧光特性发生显著改变的一类荧光蛋白。借助光激活荧光蛋白的这种特性,可以实现对活细胞、细胞器或胞内分子的时空标记和追踪。该文介绍了目前光激活荧光蛋白的性质, 并从多个方面对其应用进行了概括, 包括分子标记与动态分析、蛋白质相互作用、细胞器及细胞组分动态研究、细胞追踪以及在光激活定位显微镜中的应用等, 且对目前光激活荧光蛋白在植物分子细胞生物学中的应用进行了详细介绍。     

荧光蛋白研究进展

荧光蛋白在生物学众多研究领域中有着广泛的应用,基于荧光蛋白的分子探针和标记方法已成为活细胞或活体内动态成像研究生物大分子或细胞功能的重要工具。本文对现有荧光蛋白的种类和理化特性,及其在生物学研究中的应用进行了综述介绍。重点介绍了近年来荧光蛋白在亮度、Stokes位移、光谱改变等方面的研究进展,介绍了光转换与光活化荧光蛋白及其在超分辨荧光成像技术中的应用。最后对荧光蛋白未来的发展方向进行了展望。     

基于荧光蛋白的荧光共振能量转移探针的构建及应用

荧光共振能量转移(fluorescence resonance energy transfer,FRET)是基于荧光基团供体和荧光基团受体间偶极子–偶极子耦合作用的非辐射方式的能量传递现象。基于荧光蛋白的FRET技术已被广泛用于研究细胞信号通路中蛋白质–蛋白质活体相互作用检测、蛋白质构象变化监测以及生物探针的研制中。基于荧光蛋白的荧光共振能量转移探针使得人们可以在时间和空间层面上研究细胞信号的转导过程。该文简要介绍了四大类基于荧光蛋白的FRET生物探针的设计、研制以及其在生物信号分子检测、活细胞成像以及药物筛选中的应用和进展情况。     

代谢性标记结合二维电泳寻找O-糖基化蛋白的研究

蛋白质的O-糖基化(O-glycosylation)是一种重要的翻译后修饰,参与诸多生理和病理过程。目前,对于蛋白质的O-糖基化的研究进展仍非常缓慢,一个重要的原因就是缺乏高效的对O-糖基化蛋白进行分离和鉴定的技术。创新性地将点击化学反应、二维电泳和质谱技术结合,对寻找细胞内O-糖基化蛋白进行了技术探索性研究。首先利用代谢性标记手段,在人肝癌细胞HCCLM6培养基中加入四乙酰化叠氮半乳糖胺(Ac4GalNAz),对细胞内O-GalNAc糖基化蛋白进行标记;其次通过点击化学将炔基荧光基团连接至标记的O-糖基化蛋白的叠氮基团;应用二维电泳技术对标记蛋白进行分离,并找到13个具有荧光信号的蛋白点;最后对具有荧光信号的蛋白进行质谱鉴定,成功鉴定到7种蛋白,经软件预测后,GRP78蛋白和ANXA1蛋白均具有潜在的O-糖基化位点。这为寻找细胞或生物体中的O-糖基化蛋白奠定了基础,并为高通量筛选O-糖基化蛋白提供技术平台。     

含Dsred类似生色团的红色荧光蛋白的发展及应用

自从绿色荧光蛋白(GFP)被发现以来,荧光蛋白在生物医学领域已经成为一种重要的荧光成像工具．随着红色荧光蛋白DsRed的出现,各种优化的DsRed突变体和远红荧光蛋白也不断涌现．其中荧光蛋白生色团的形成机制对改建更优的荧光蛋白变种影响很大,对于红色荧光蛋白而言,大多数的红色荧光蛋白的生色团类型为DsRed类似生色团,在此基础上又出现了Far-red DsRed类似生色团．目前,含DsRed类似生色团的荧光蛋白主要有单体红色荧光蛋白、光转换荧光蛋白、斯托克斯红移蛋白、荧光计时器等．这些优化的荧光蛋白作为分子探针可以实现对活细胞、细胞器或胞内分子的时空标记和追踪,已经在生物工程学、细胞生物学、基础医学领域得到广泛应用．本文综述了含DsRed类似生色团的荧光蛋白的研究进展及其应用,以及由此发展起来的远红荧光蛋白在活体显微成像技术中的应用,并展望了荧光探针技术研究的新方向．     

绿色荧光蛋白及其在细胞生物研究中的应用

绿色荧光蛋白(green fluorescent protein,GFP)作为一种新型的标记蛋白,已被广泛的应用。它发出的荧光稳定,检测简单,结果真实可靠。GFP可对活细胞的生理过程进行监控,并且可以用于活细胞中蛋白质分子的定位及动力学研究。其特有的生物化学性质使其在细胞生物学和分子生物学领域有着广泛的应用前景。     

绿色荧光蛋白及其在细胞生物学研究中的应用

绿色荧光蛋白(green fluorescent protein,GFP)作为一种新型的标记蛋白,已被广泛的应用.它发出的荧光稳定,检测简单,结果真实可靠.GFP可对活细胞的生理过程进行监控,并且可以用于活细胞中蛋白质分子的定位及动力学研究.其特有的生物化学性质使其在细胞生物学和分子生物学领域有着广泛的应用前景.     

BK通道在细胞膜上的单分子定位及空间分布

目的：研究大电导、钙离子和电压激活的钾离子通道（BK通道）在HEK293细胞膜上的单分子定位及其总体空间分布情况。方法：分别用mEos2、Dronpa等荧光蛋白标记BK通道的α亚基和辅助性β2亚基,将这些质粒在HEK293细胞内瞬时转染以表达通道蛋白,然后用激光共聚焦荧光显微成像、全内反射荧光显微成像、光敏定位荧光成像等技术观察BK通道的亚细胞定位及单分子分布,并用电生理实验技术检测荧光蛋白对BK通道有影响。结果：激光共聚焦荧光显微成像和全内反射荧光显微成像技术只能在亚细胞水平定位通道蛋白,BK通道在细胞膜上聚集并形成不规则的蛋白簇,它的仅亚基和β2亚基在细胞膜上完全共定位;光敏定位荧光成像技术成功定位BK通道蛋白簇里面的单分子,虽然α和β2亚基紧紧靠在一起,它们之间依然存在空间距离;BK通道的质膜表达和功能特性不受荧光蛋白的影响。结论：BK通道蛋白簇里面包含大量的α和β2亚基的蛋白单分子,它们紧密地聚集在一起,但是并没有完全共定位,在分子水平上揭示了BK通道α和p亚基功能耦合的结构基础,为以后研究大分子蛋白质间的相互作用机制提供了很好的分子模型,光敏定位荧光成像技术作为一种全新的单分子荧光成像手段,在基因表达、信号通路、蛋白质相互作用等许多重要生命活动的研究中发挥重要作用。     

绿色荧光蛋白在蛋白质研究中的应用

绿色荧光蛋白（green fluorescent protein,GFP）自发现以来,由于具有自发荧光等特性,在分子生物学和细胞生物学领域得到广泛应用。GFP作为一种报道分子,在研究蛋白质相互作用和构象变化、检测蛋白质表达、蛋白质和细胞荧光示踪中,起到了重要的作用。该文通过对绿色荧光蛋白特性的分析．介绍其作为荧光标记在蛋白质研究中的应用,并展望进一步的研究前景。     

Multicolor protein labeling in living cells using mutant β-lactamase-tag technology

Protein labeling techniques using small molecule probes have become important as practical alternatives to the use of fluorescent proteins (FPs) in live cell imaging. These labeling techniques can be applied to more sophisticated fluorescence imaging studies such as pulse-chase imaging. Previously, we reported a novel protein labeling system based on the combination of a mutant β-lactamase (BL-tag) with coumarin-derivatized probes and its application to specific protein labeling on cell membranes. In this paper, we demonstrated the broad applicability of our BL-tag technology to live cell imaging by the development of a series of fluorescence labeling probes for this technology, and the examination of the functions of target proteins. These new probes have a fluorescein or rhodamine chromophore, each of which provides enhanced photophysical properties relative to coumarins for the purpose of cellular imaging. These probes were used to specifically label the BL-tag protein and could be used with other small molecule fluorescent probes. Simultaneous labeling using our new probes with another protein labeling technology was found to be effective. In addition, it was also confirmed that this technology has a low interference with respect to the functions of target proteins in comparison to GFP. Highly specific and fast covalent labeling properties of this labeling technology is expected to provide robust tools for investigating protein functions in living cells, and future applications can be improved by combining the BL-tag technology with conventional imaging techniques. The combination of probe synthesis and molecular biology techniques provides the advantages of both techniques and can enable the design of experiments that cannot currently be performed using existing tools.     

Tracking single Kinesin molecules in the cytoplasm of mammalian cells

Understanding dynamic cellular processes requires precise knowledge of the distribution, transport, and interactions of individual molecules in living cells. Despite recent progress in in vivo imaging, it has not been possible to express and directly track single molecules in the cytoplasm of live cells. Here, we overcome these limitations by combining fluorescent protein-labeling with high resolution total internal reflection fluorescence microcopy, using the molecular motor Kinesin-1 as model system. First, we engineered a three-tandem monomeric Citrine tag for genetic labeling of individual molecules and expressed this motor in COS cells. Detailed analysis of the quantized photobleaching behavior of individual fluorescent spots demonstrates that we are indeed detecting single proteins in the cytoplasm of live cells. Tracking the movement of individual cytoplasmic molecules reveals that individual Kinesin-1 motors in vivo move with an average speed of 0.78 +/- 0.11 microm/s and display an average run length of 1.17 +/- 0.38 microm, which agrees well with in vitro measurements. Thus, Kinesin-1's speed and processivity are not upregulated or hindered by macromolecular crowding. Second, we demonstrate that standard deviation maps of the fluorescence intensity computed from single molecule image sequences can be used to reveal important physiological information about infrequent cellular events in the noisy fluorescence background of live cells. Finally, we show that tandem fluorescent protein tags enable single-molecule, in vitro analyses of extracted, mammalian-expressed proteins. Thus, by combining direct genetic labeling and single molecule imaging in vivo, our work establishes an important new biophysical method for observing single molecules expressed and localized in the mammalian cytoplasm.     

A general method for the covalent labeling of fusion proteins with small molecules in vivo

Characterizing the movement, interactions, and chemical microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic molecules capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small molecule to a protein, the formation of stable complexes between biarsenical compounds and peptides containing cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.     

Total internal reflection fluorescence microscopy for single-molecule imaging in living cells

Marvelous background rejection in total internal reflection fluorescence microscopy (TIR-FM) has made it possible to visualize single-fluorophores in living cells. Cell signaling proteins including peptide hormones, membrane receptors, small G proteins, cytoplasmic kinases as well as small signaling compounds have been conjugated with single chemical fluorophore or tagged with green fluorescent proteins and visualized in living cells. In this review, the reasons why single-molecule analysis is essential for studies of intracellular protein systems such as cell signaling system are discussed, the instrumentation of TIR-FM for single-molecule imaging in living cells is explained, and how single molecule visualization has been used in cell biology is illustrated by way of two examples: signaling of epidermal growth factor in mammalian cells and chemotaxis of Dictyostelium amoeba along a cAMP gradient. Single-molecule analysis is an ideal method to quantify the parameters of reaction dynamics and kinetics of unitary processes within intracellular protein systems. Knowledge of these parameters is crucial for the understanding of the molecular mechanisms underlying intracellular events, thus single-molecule imaging in living cells will be one of the major technologies in cellular nanobiology.     

Labeling neurons in vivo for morphological and functional studies

Increasingly sophisticated strategies for labeling cells in vivo are providing unprecedented opportunities to study neurons in living animals. Transgenic expression of genetically encoded reporters enables us to monitor changes in neuronal activity in response to sensory stimuli, and the labeling of single neurons with fluorescent proteins allows the dynamics of neuronal connectivity to be observed in transgenic animals over periods ranging from minutes to months. Advances in transient labeling techniques such as viral infection and electroporation provide a rapid means by which to analyze neuronal gene function in vivo. These new approaches to labeling, manipulating and imaging neurons in intact organisms are transforming the way in which the nervous system is studied.     

Fluorescent labels for proteomics and genomics

Fluorescent labeling reagents are an essential component of a huge industry built on sensitive fluorescence detection. This technology has grown over 30 years and is in some ways mature. Excellent labeling reagents with close to maximum theoretical brightness are available in many different colors. Large fluorescent proteins like phycobiliproteins are also widely used that are exceedingly bright. Other fluorescent proteins like the GFP family can be obtained for creating genetically encoded protein labels in living cells. A new 'solid state' quantum dot technology is being exploited for large-scale multiparameter labeling. This technology provides the 'ultimate' photostable labeling reagent. Still, there are advances to be made. Not available is the ultimate tool kit of low molecular weight, strongly light absorbing, photostable labels with narrow emission bands ranging from the UV to the IR.     

Development of an ON/OFF switchable fluorescent probe targeting His tag fused proteins in living cells

The fluorescent labeling of target proteins is useful for analyzing their functions and localization in cells, and several fluorescent probes have been developed. However, the fusion of tags such as green fluorescent protein (GFP) to target proteins occasionally affects their functions and/or localization in living cells. Therefore, an imaging method that uses short peptide tags such as hexa-histidine (the His tag) has been attracting increasing attention. Few studies have investigated ON/OFF switchable fluorescent probes for intracellular His-tagged proteins. We herein developed a novel ON/OFF switchable probe for imaging targeted intracellular proteins fused with a CH6 tag, which is composed of one cysteine residue and six histidine residues.     

Dynamic Superresolution Imaging of Endogenous Proteins on Living Cells at Ultra-High Density

Versatile superresolution imaging methods, able to give dynamic information of endogenous molecules at high density, are still lacking in biological science. Here, superresolved images and diffusion maps of membrane proteins are obtained on living cells. The method consists of recording thousands of single-molecule trajectories that appear sequentially on a cell surface upon continuously labeling molecules of interest. It allows studying any molecules that can be labeled with fluorescent ligands including endogenous membrane proteins on living cells. This approach, named universal PAINT (uPAINT), generalizes the previously developed point-accumulation-for-imaging-in-nanoscale-topography (PAINT) method for dynamic imaging of arbitrary membrane biomolecules. We show here that the unprecedented large statistics obtained by uPAINT on single cells reveal local diffusion properties of specific proteins, either in distinct membrane compartments of adherent cells or in neuronal synapses.     

RESOLFT Nanoscopy of Fixed Cells Using a Z-Domain Based Fusion Protein for Labelling

RESOLFT super-resolution microscopy allows subdiffraction resolution imaging of living cells using low intensities of light. It relies on the light-driven switching of reversible switchable fluorescent proteins (RSFPs). So far, RESOLFT imaging was restricted to living cells, because chemical fixation typically affects the switching characteristics of RSFPs. In this study we created a fusion construct (FLASR) consisting of the RSFP rsEGFP2 and the divalent form of the antibody binding Z domain from protein A. FLASR can be used analogous to secondary antibodies in conventional immunochemistry, facilitating simple and robust sample preparation. We demonstrate RESOLFT super-resolution microscopy on chemically fixed mammalian cells. The approach may be extended to other super-resolution approaches requiring fluorescent proteins in an aqueous environment.     

Imaging proteins inside cells with fluorescent tags

Watching biological molecules provides clues to their function and regulation. Some of the most powerful methods of labeling proteins for imaging use genetically encoded fluorescent fusion tags. There are four standard genetic methods of covalently tagging a protein with a fluorescent probe for cellular imaging. These use (i) autofluorescent proteins, (ii) self-labeling enzymes, (iii) enzymes that catalyze the attachment of a probe to a target sequence, and (iv) biarsenical dyes that target tetracysteine motifs. Each of these techniques has advantages and disadvantages. In this review, we cover new developments in these methods and discuss practical considerations for their use in imaging proteins inside living cells.