Visualization of growth signal transduction cascades in living cells with genetically encoded probes based on Förster resonance energy transfer

Fluorescence probes based on the principle of Förster resonance energy transfer (FRET) have shed new light on our understanding of signal transduction cascades. Among them, unimolecular FRET probes containing fluorescence proteins are rapidly increasing in number because these genetically encoded probes can be easily loaded into living cells and allow simple acquisition of FRET images. We have developed probes for small GTPases, tyrosine kinases, serine–threonine kinases and phosphoinositides. Images obtained with these probes have revealed that membrane protrusions such as nascent lamellipodia or neurites provide an active signalling platform in the growth factor-stimulated cells.     

Non fitting based FRET–FLIM analysis approaches applied to quantify protein–protein interactions in live cells

New imaging methodologies in quantitative fluorescence microscopy and nanoscopy have been developed in the last few years and are beginning to be extensively applied to biological problems, such as the localization and quantification of protein interactions. Fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) is currently employed not only in biophysics or chemistry but also in bio-medicine, thanks to new advancements in technology and also new developments in data treatment. FRET–FLIM can be a very useful tool to ascertain protein interactions occurring in single living cells. In this review, we stress the importance of increasing the acquisition speed when working in vivo employing Time-Domain FLIM. The development of the new mathematical-based non-fitting methods allows the determining of the fraction of interacting donor without the requirement of high count statistics, and thus allows the performing of high speed acquisitions in FRET–FLIM to still be quantitative.     

Concerted release of substrate domains from GroEL by ATP is demonstrated with FRET

The chaperonin GroEL assists protein folding by undergoing ATP-induced conformational changes that are concerted within each of its two back-to-back stacked rings. Here we examined whether concerted allosteric switching gives rise to all-or-none release and folding of domains in a chimeric fluorescent protein substrate, CyPet-YPet. Using this substrate, it was possible to determine the folding yield of each domain from its intrinsic fluorescence and that of the entire chimera by measuring Förster resonance energy transfer between the two domains. Hence, it was possible to determine whether release of one domain is accompanied by release of the other domain (concerted mechanism), or whether their release is not coupled. Our results show that the chimera's release tends to be concerted when folding is assisted by a wild-type GroEL variant, but not when assisted by the F44W/D155A mutant that undergoes a sequential allosteric switch. A connection between the allosteric mechanism of this molecular machine and its biological function in assisting folding is thus established.     

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using Förster resonance energy transfer (FRET)^1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.     

Efficiency of Resonance Energy Transfer in Homo-Oligomeric Complexes of Proteins

A theoretical model is proposed for the apparent efficiency of fluorescence (Förster) resonance energy transfer (FRET) in mixtures of free monomers and homo-oligomeric protein complexes of uniform size. The model takes into account possible pathways for transfer of optical excitations from single donors to multiple acceptors and from multiple donors (non-simultaneously) to single acceptors. This necessary departure from the standard theory has been suggested in the literature, but it has only been successfully implemented for a few particular cases, such as for particular geometries of the oligomers. The predictions of the present theoretical model differ significantly from those of the standard theory, with the exception of the case of dimers, for which agreement is observed. This model therefore provides new insights into the FRET behavior of oligomers comprising more than two monomers, and also suggests means for determining the size of oligomeric protein complexes as well as the proportion of associated and unassociated monomers.     

The Ever Changing Moods of Calmodulin: How Structural Plasticity Entails Transductional Adaptability

The exceptional versatility of calmodulin (CaM) three-dimensional arrangement is reflected in the growing number of structural models of CaM/protein complexes currently available in the Protein Data Bank (PDB) database, revealing a great diversity of conformations, domain organization, and structural responses to Ca^2 +. Understanding CaM binding is complicated by the diversity of target proteins sequences. Data mining of the structures shows that one face of each of the eight CaM helices can contribute to binding, with little overall difference between the Ca^2 + loaded N- and C-lobes and a clear prevalence of the C-lobe low Ca^2 + conditions. The structures reveal a remarkable variety of configurations where CaM binds its targets in a preferred orientation that can be reversed and where CaM rotates upon Ca^2 + binding, suggesting a highly dynamic metastable relation between CaM and its targets. Recent advances in structure–function studies and the discovery of CaM mutations being responsible for human diseases, besides expanding the role of CaM in human pathophysiology, are opening new exciting avenues for the understanding of the how CaM decodes Ca^2 +-dependent and Ca^2 +-independent signals.     

Single-molecule Förster resonance energy transfer studies of RNA structure, dynamics and function

Single-molecule fluorescence microscopy experiments on RNA molecules brought to light the highly complex dynamics of key biological processes, including RNA folding, catalysis of ribozymes, ligand sensing of riboswitches and aptamers, and protein synthesis in the ribosome. By using highly advanced biophysical spectroscopy techniques in combination with sophisticated biochemical synthesis approaches, molecular dynamics of individual RNA molecules can be observed in real time and under physiological conditions in unprecedented detail that cannot be achieved with bulk experiments. Here, we review recent advances in RNA folding and functional studies of RNA and RNA-protein complexes addressed by using single-molecule Förster (fluorescence) resonance energy transfer (smFRET) technique.     

盐离子作用下组蛋白变体H2A.Z增强核小体结构的稳定性

真核生物染色质的基本结构组成单元是核小体,基因组DNA被压缩在染色质中,核小体的存在通常会抑制转录、复制、修复和重组等发生在DNA模板上的生物学过程。组蛋白变体H2A.Z可以调控染色质结构进而影响基因的转录过程,但其详细的调控机制仍未研究清楚。为了比较含有组蛋白变体H2A.Z的核小体和常规核小体在盐离子作用下的稳定性差异,本文采用Förster共振能量转移的方法检测氯化钠、氯化钾、氯化锰、氯化钙、氯化镁等离子对核小体的解聚影响。实验对Widom 601 DNA序列进行双荧光Cy3和Cy5标记,通过荧光信号值的变化来反映核小体的解聚变化。Förster共振能量转移检测结果显示:在氯化钠、氯化钾、氯化锰、氯化钙和氯化镁作用下,含有组蛋白变体H2A.Z的核小体解聚速度相比于常规核小体要慢,且氯化钙、氯化锰和氯化镁的影响更明显。电泳分析结果表明,在75℃条件下含有组蛋白变体H2A.Z的核小体的解聚速率明显低于常规核小体。采用荧光热漂移检测(fluorescence thermal shift analysis , FTS)进一步分析含有组蛋白变体H2A.Z核小体的稳定性,发现两类核小体的荧光信号均呈现2个明显的增长期,含有组蛋白变体H2A.Z核小体的第1个荧光信号增速期所对应的温度明显高于常规核小体,表明核小体中H2A.Z/H2B二聚体的解聚变性温度要高于常规的H2A/H2B二聚体,含有组蛋白变体H2A.Z核小体的热稳定性高。研究结果均表明,含有组蛋白变体H2A.Z的核小体的结构比常规核小体的结构稳定。     

FRET Imaging in Three-dimensional Hydrogels

Imaging of Förster resonance energy transfer (FRET) is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.     

Proteasome inhibition can induce an autophagy-dependent apical activation of caspase-8

Antiapoptotic Bcl-2 family proteins are often highly expressed in chemotherapy-resistant cancers and impair mitochondrial outer membrane permeabilisation (MOMP), an important requirement for caspase activation via the intrinsic apoptosis pathway. Interestingly, although Bcl-2 overexpression in HeLa cervical cancer cells abrogated caspase processing in response to intrinsic apoptosis induction by staurosporine, tunicamycin or etoposide, residual caspase processing was observed following proteasome inhibition by bortezomib ([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid), epoxomicin (N-acetyl-N-methyl-lisoleucyl-L-isoleucyl-N-[(1S)-3-methyl-1-[[(2R)-2-methyloxiranyl]carbonyl]butyl]-L-threoninamide) or MG-132 (N-(benzyloxycarbonyl)leucinylleucinylleucinal). Similar responses were found in Bcl-2-overexpressing H460 NSCLC cells and Bax/Bak-deficient mouse embyronic fibroblasts. Mild caspase processing resulted in low DEVDase activities, which were MOMP independent and persisted for long periods without evoking immediate cell death. Surprisingly, depletion of caspase-3 and experiments in caspase-7-depleted MCF-7-Bcl-2 cells indicated that the DEVDase activity did not originate from effector caspases. Instead, Fas-associated death domain (FADD)-dependent caspase-8 activation was the major contributor to the slow, incomplete substrate cleavage. Caspase-8 activation was independent of death ligands, but required the induction of autophagy and the presence of Atg5. Depletion of XIAP or addition of XIAP-antagonising peptides resulted in a switch towards efficient apoptosis execution, suggesting that the requirement for MOMP was bypassed by activating the caspase-8/caspase-3 axis. Combination treatments of proteasome inhibitors and XIAP antagonists therefore represent a promising strategy to eliminate highly resistant cancer cells, which overexpress antiapoptotic Bcl-2 family members.     

Microarray analysis of protein-protein interactions based on FRET using subnanosecond-resolved fluorescence lifetime imaging

The detection of protein-protein binding on microarrays using the fluorescence lifetime as a dynamic analytical parameter was investigated in a model system. The assay is based on F?rster resonance energy transfer (FRET) and carried out with biotinylated Bovine Serum Albumin and streptavidin, labeled with the commonly used microarray dyes Alexa 555 and Alexa 647, respectively. This efficient FRET donor/acceptor pair was employed in a competitive assay format on three different microarray surfaces. The fluorescence was excited by 200ps laser pulses from a mode-locked and cavity-dumped argon-ion laser, adapted to an intensified CCD camera as detection unit allowing time resolution with subnanosecond precision. Lifetime maps were recorded according to the Rapid Lifetime Determination (RLD) scheme. Interaction between the proteins could clearly be detected on all formats and resulted in almost complete quenching on CEL Epoxy surfaces upon addition of excess streptavidin labeled the FRET acceptor dye. In this case, the fluorescence lifetimes dropped by 90%, whereas on ARChip Epoxy and ARChip Gel the reduction was 54% and 47%, respectively. Good linearity of the quenching curve was obtained in all cases. The method is applicable to all types of protein interaction analysis on microarrays, particularly in cases where evaluation of fluorescence intensity is prone to erroneous results and a more robust parameter is required.     

Using Co-Cultures Expressing Fluorescence Resonance Energy Transfer Based Protein Biosensors to Simultaneously Image Caspase-3 and Ca<Superscript>2+</Superscript> Signaling

Fluorescence resonance energy transfer (FRET)-based protein biosensors allow the spatial and temporal imaging of signaling events in living cells. However, the simultaneous correlation of multiple events of a signaling pathway is hindered by the spectral cross-talk between fluorescent proteins. Here, we show, for signaling pathways that progress synchronously, multiple events can be correlated by using co-cultures expressing different FRET-based protein biosensors. As a demonstration, we investigated the simultaneous caspase-3 and Ca²⁺ signaling events involved in cell death of COS-7 cells induced by 10 mM H₂O₂. Interestingly, this H₂O₂ stimulus induced synchronous caspase-3 activation and Ca²⁺ signaling. In parallel to caspase-3 activation, cytosolic Ca²⁺ concentration, [Ca²⁺]_c, gradually rises to its peak and then slowly drops. As cell shrinkage and rounding ensues, [Ca²⁺]_c again gradually rises to its peak and then reaches a plateau. These observations reveal the relative timing and location of these signaling events in cell death induced by this stimulus of H₂O₂. Finally, our approach offers an exciting opportunity for spatial and temporal imaging of multiple events in a signaling pathway in living cells.     

在活细胞中应用FRET技术研究TGFβ/TβRI信号传导

转移生长因子β(TGFβ)信号传导通路参与调节细胞的增殖、分化、凋亡、细胞迁移等一系列细胞过程,与骨代谢疾病的发病机制密切相关.本研究根据荧光共振能量转移(FRET)技术原理,构建包含CFP-TβRI-YFP融合蛋白的TβRI生物传感器,转染293T细胞,观察转染效率.以TGFβ1为诱导剂,激活TGFβ/TβRI信号传导通路,在活细胞生理条件下,动态监测TβRI生物传感器的FRET效应.结果表明,成功构建了TβRI生物传感器,转染细胞效率达50%,在TGFβ1诱导刺激6min后,FRET效率增加并达到最大值,该过程经历9 min后,随时间的延长,FRET效率下降.研究结果表明:在活细胞生理条件下,TGFβ1/TβRI信号转导过程存在一定的时间特异性.     

Physical responses of bacterial chemoreceptors

Chemoreceptors of the bacterium Escherichia coli are thought to form trimers of homodimers that undergo conformational changes upon ligand binding and thereby signal a cytoplasmic kinase. We monitored the physical responses of trimers in living cells lacking other chemotaxis proteins by fluorescently tagging receptors and measuring changes in fluorescence anisotropy. These changes were traced to changes in energy transfer between fluorophores on different dimers of a trimer: attractants move these fluorophores farther apart, and repellents move them closer together. These measurements allowed us to define the responses of bare receptor oligomers to ligand binding and compare them to the corresponding response in kinase activity. Receptor responses could be fit by a simple two-state model in which receptor dimers are in either active or inactive conformations, from which energy bias and dissociation constants could be estimated. Comparison with responses in kinase-activity indicated that higher-order interactions are dominant in receptor clusters.