RNA mimics of green fluorescent protein

Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here, we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex, termed Spinach, resembles enhanced GFP and emits a green fluorescence comparable in brightness with fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.     

Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells

Many proteins associated with the plasma membrane are known to partition into submicroscopic sphingolipid- and cholesterol-rich domains called lipid rafts, but the determinants dictating this segregation of proteins in the membrane are poorly understood. We suppressed the tendency of Aequorea fluorescent proteins to dimerize and targeted these variants to the plasma membrane using several different types of lipid anchors. Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts. Thus the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.     

A vital stain for the Golgi apparatus

The Golgi complex, a membranous organelle with important functions in membrane traffic and macromolecular synthesis, has been stained in living cells with a fluorescent sphingolipid. Cells were first incubated with liposomes containing N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl sphingosine (C6-NBD-ceramide), or with a bovine serum albumin complex of the fluorescent lipid, and then examined by fluorescence microscopy. An intensely fluorescent perinuclear structure was identified as the Golgi apparatus by its colocalization with known Golgi markers in fixed cells. C6-NBD-ceramide was used to observe the morphology of the Golgi apparatus in living cells in the presence or absence of monensin or Colcemid, and during mitosis. In all cases, C6-NBD-ceramide revealed a Golgi apparatus in the living cell that was identical to that obtained with conventional procedures that require fixation.     

绿色荧光蛋白在植物上应用研究进展

绿色荧光蛋白(green fluorescent protein,GFP)是一类存在于海洋生物多管水母、水螅和珊瑚等腔肠动物体内的一种功能独特的生物发光蛋白。能够自身催化形成生色团并在蓝光或紫外光激发下发出绿色荧光,能在活细胞内稳定表达。随着进一步对绿色荧光蛋白结构及其发光特性的研究,以及对绿色荧光蛋白基因的成功克隆,对多种新型绿色荧光蛋白的成功改造,使对多种目标进行实时监测成为可能。文中综述了绿色荧光蛋白的发展过程、改进及应用的研究进展。     

Receptor-mediated activation of heterotrimeric G-proteins in living cells

Receptor-mediated activation of heterotrimeric GTP-binding proteins (G-proteins) was visualized in living Dictyostelium discoideum cells by monitoring fluorescence resonance energy transfer (FRET) between alpha- and beta- subunits fused to cyan and yellow fluorescent proteins. The G-protein heterotrimer rapidly dissociated and reassociated upon addition and removal of chemoattractant. During continuous stimulation, G-protein activation reached a dose-dependent steady-state level. Even though physiological responses subsided, the activation did not decline. Thus, adaptation occurs at another point in the signaling pathway, and occupied receptors, whether or not they are phosphorylated, catalyze the G-protein cycle. Construction of similar energy-transfer pairs of mammalian G-proteins should enable direct in situ mechanistic studies and applications such as drug screening and identifying ligands of newly found G-protein-coupled receptors.     

Fluorescence imaging of cellular metabolites with RNA

Genetically encoded sensors are powerful tools for imaging intracellular metabolites and signaling molecules. However, developing sensors is challenging because they require proteins that undergo conformational changes upon binding the desired target molecule. We describe an approach for generating fluorescent sensors based on Spinach, an RNA sequence that binds and activates the fluorescence of a small-molecule fluorophore. We show that these sensors can detect a variety of different small molecules in vitro and in living cells. These RNAs constitute a versatile approach for fluorescence imaging of small molecules and have the potential to detect essentially any cellular biomolecule.     

The fluorescent toolbox for assessing protein location and function

Advances in molecular biology, organic chemistry, and materials science have recently created several new classes of fluorescent probes for imaging in cell biology. Here we review the characteristic benefits and limitations of fluorescent probes to study proteins. The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy. Small organic fluorescent dyes, nanocrystals ("quantum dots"), autofluorescent proteins, small genetic encoded tags that can be complexed with fluorochromes, and combinations of these probes are highlighted.     

果蝇生殖包囊固定染色后的荧光特性

[目的]探索果蝇卵巢及生殖包囊经过常规固定和染色后的荧光特性,为荧光组织学的发展提供第一手资料。[方法]以黑腹果蝇卵巢为试材,用Bouin氏液固定,石蜡切片后分别用HE、苏木精、伊红染色,在明场（白光）、绿色、蓝色和紫外激发光下观察、拍照。[结果]3种方法染色后,再经不同激发光激发,生殖细胞和体细胞可发出不同颜色的荧光,细胞中脂类、核酸及蛋白质也可分别发出各自特异性的荧光。[结论]常规染色剂可赋予生殖细胞和体细胞各自不同的荧光特性,也可赋予细胞内不同物质成分各自不同的荧光特性。     

果蝇生殖包囊固定染色后的荧光特性（摘要）

[目的]探索果蝇卵巢及生殖包囊经过常规固定和染色后的荧光特性,为荧光组织学的发展提供第一手资料。[方法]以黑腹果蝇卵巢为试验材料,用Bouin氏液固定,石蜡切片后分别用HE、苏木精、伊红染色,分别在明场（白光）、绿色、蓝色和紫外激发光下观察、拍照。[结果]3种方法染色后,再经不同激发光激发,生殖细胞和体细胞可发出不同颜色的荧光,细胞中脂类、核酸及蛋白质也可分别发出各自特异性的荧光。[结论]常规染色剂可以分别赋予生殖细胞和体细胞各自不同的荧光特性,也可赋予细胞内不同物质成份各自不同的荧光特性。     

Proteome half-life dynamics in living human cells

Cells remove proteins by two processes: degradation and dilution due to cell growth. The balance between these basic processes is poorly understood. We addressed this by developing an accurate and noninvasive method for measuring protein half-lives, called "bleach-chase," that is applicable to fluorescently tagged proteins. Assaying 100 proteins in living human cancer cells showed half-lives that ranged between 45 minutes and 22.5 hours. A variety of stresses that stop cell division showed the same general effect: Long-lived proteins became longer-lived, whereas short-lived proteins remained largely unaffected. This effect is due to the relative strengths of degradation and dilution and suggests a mechanism for differential killing of rapidly growing cells by growth-arresting drugs. This approach opens a way to understand proteome half-life dynamics in living cells.     

Cell biology. Color-changing protein times gene activity

For years, researchers trying to capture the bustling activity of genes in living cells have mostly had to make do with snapshots, which clearly fell short of the mark. But now their frustration may be over. On page 1585, a team describes a new fluorescent protein that turns bright green when it is first made, then changes to red over several hours--providing the ability to witness how genes alter their activities over time.     

Light microscopy techniques for live cell imaging

Since the earliest examination of cellular structures, biologists have been fascinated by observing cells using light microscopy. The advent of fluorescent labeling technologies plus the plethora of sophisticated light microscope techniques now available make studying dynamic processes in living cells almost commonplace. For anyone new to this area, however, it can be daunting to decide which techniques or equipment to try. Here, we aim to give a brief overview of the main approaches to live cell imaging, with some mention of their pros and cons.     

Probing gene expression in live cells, one protein molecule at a time

We directly observed real-time production of single protein molecules in individual Escherichia coli cells. A fusion protein of a fast-maturing yellow fluorescent protein (YFP) and a membrane-targeting peptide was expressed under a repressed condition. The membrane-localized YFP can be detected with single-molecule sensitivity. We found that the protein molecules are produced in bursts, with each burst originating from a stochastically transcribed single messenger RNA molecule, and that protein copy numbers in the bursts follow a geometric distribution. The quantitative study of low-level gene expression demonstrates the potential of single-molecule experiments in elucidating the workings of fundamental biological processes in living cells.     

Defining lipid transport pathways in animal cells

A new technique for studying the metabolism and intracellular transport of lipid molecules in living cells based on the use of fluorescent lipid analogs is described. The cellular processing of various intermediates (phosphatidic acid and ceramide) and end products (phosphatidylcholine and phosphatidylethanolamine) in lipid biosynthesis is reviewed and a working model for compartmentalization during lipid biosynthesis is presented.     

Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations

Heterozygous mutations encoding abnormal forms of the death receptor Fas dominantly interfere with Fas-induced lymphocyte apoptosis in human autoimmune lymphoproliferative syndrome. This effect, rather than depending on ligand-induced receptor oligomerization, was found to stem from ligand- independent interaction of wild-type and mutant Fas receptors through a specific region in the extracellular domain. Preassociated Fas complexes were found in living cells by means of fluorescence resonance energy transfer between variants of green fluorescent protein. These results show that formation of preassociated receptor complexes is necessary for Fas signaling and dominant interference in human disease.     

水稻条纹病毒编码的NS2蛋白的亚细胞定位分析

通过RT-PCR克隆获得水稻条纹病毒（RSV）编码的蛋白NS2和拟南芥柯浩体蛋白Atcoilin的cDNA,将两者分别与表达载体pEarley102（带青色荧光蛋白,CFP）和pEarley101（带黄色荧光蛋白,YFP）同源重组.将构建好的重组载体分别转化到农杆菌EHA105中,通过农杆菌接种法将两者共同注射到本氏烟叶片表皮细胞中进行表达.在共聚焦显微镜下观察,NS2-CFP与Atcoilin-YFP能重叠在一起,表明RSV NS2定位于柯浩体.     

Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking

Semiconductor quantum dots (QDs) are nanometer-sized fluorescent probes suitable for advanced biological imaging. We used QDs to track individual glycine receptors (GlyRs) and analyze their lateral dynamics in the neuronal membrane of living cells for periods ranging from milliseconds to minutes. We characterized multiple diffusion domains in relation to the synaptic, perisynaptic, or extrasynaptic GlyR localization. The entry of GlyRs into the synapse by diffusion was observed and further confirmed by electron microscopy imaging of QD-tagged receptors.     

TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity

Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.