Fluorescent Protein Labeling Research Articles

Fluorescent Labeling of Proteins.

Many single-molecule experimental techniques exploit fluorescence as a tool to investigate conformational dynamics, molecular interactions, or track the movement of proteins in order to gain insight into their biological functions. A prerequisite to these experimental approaches is to graft one or more fluorophores on the protein of interest with the desired photophysical properties. Here, we describe procedures for efficient methods used to covalently attach fluorophores to proteins. Alternative direct and indirect labeling strategies are also described.

Highly chromophoric Cy5-methionine for N-terminal fluorescent tagging of proteins in eukaryotic translation systems

Despite significant advances on fluorescent labeling of target proteins to study their structural dynamics and function, there has been need for labeling with high quantum yield ensuring high sensitivity and selectivity without sacrificing the biological function of the protein. Here as a technical advancement over non-canonical amino acid incorporation, we provided a conceptual design of the N-terminal fluorescent tagging of proteins. Cy5-labeled methionine (Cy5-Met) was chemically synthesized, and then the purified Cy5-Met was coupled with synthetic human initiator tRNA by methionine tRNA synthetase. Cy5-Met-initiator tRNA (Cy5-Met-tRNAi) was purified and transfected into HeLa cells with HIV-Tat plasmid, resulting in an efficient production of Cy5-labeled HIV-Tat protein. Based on the universal requirement in translational initiation, the approach provides co-translational incorporation of N-terminal probe to a repertoire of proteins in the eukaryote system. This methodology has potential utility in the single molecule analysis of human proteins in vitro and in vivo for addressing to their complex biological structural and functional dynamics.

Sca-1 expression level identifies quiescent hematopoietic stem and progenitor cells

Blood cell generation depends on continuous cellular output by the sequential hierarchy of hematopoietic stem cell (HSC) and progenitor populations that all contain quiescent and actively cycling cells. Hematopoietic stem and progenitor cells (HSPCs) express the surface molecule Stem cell antigen 1 (SCA-1/LY6A). Using histone 2B-red fluorescent fusion protein label retention and cell-cycle reporter mice, we demonstrate that high SCA-1 expression (SCA-1hi) identifies not only quiescent HSCs but quiescent cells on all hierarchical levels within the lineage-SCA-1+KIT+ (LSK) population. Each transplanted SCA-1hi HSPC population also displayed self-renewal potential superior to that of the respective SCA-1lo population. SCA-1 expression is inducible by type I interferon (IFN). We show, however, that quiescence and high self-renewal capacity of cells with brighter SCA-1 expression at steady state were independent of type I IFN signaling. We conclude that SCA-1 expression levels can be used to prospectively isolate functionally heterogeneous HSPC subpopulations.

Fluorescent Labeling of Protein Using Blue-Emitting 8-Amino-BODIPY Derivatives.

8-Amino-BODIPY (boron-dipyrromethane) dyes show bright blue fluorescence. Disclosed here are synthesis and characterization of the photophysical properties of a series of functionalized 8-Amino-BODIPY (BP1-4) for protein labeling. The compact structure and solvent-insensitive absorption property of the dye are desirable features for protein labeling. For the model protein, bovine serum albumin (BSA), the labeling proceeds under mild condition via amide bond formation or thiol-ene conjugation with maintaining the bright blue fluorescence. The chromatography and mass spectroscopy analysis clearly support the labeling of the BODIPY dye on the BSA. The protein labeling with blue-emitting BODIPY would be applicable for studying protein dynamics and fluorescence resonance energy transfer (FRET) with intrinsic biomolecules.

Murine Supraspinatus Tendon Detachment and Repair Model Augmented With Tendon-Derived, Activated Endothelial Cells: A New Concept in Biologic Enhancement of Tendon-to-Bone Healing

Objectives:Biologic interventions are being increasingly used to improve outcome of rotator cuff repair. Individual growth factors and stem cell-based augmentations are among the most popular approaches. In this study we investigated a novel approach in which tendon-derived, activated endothelial cells (tAECs) are used as a source of tissue-specific growth factors to stimulate tendon intrinsic stem cell niche in our established murine model of microsurgical supraspinatus tendon (SST) detachment and repair.Methods:Study protocol was approved by the IACUC. In stage 1, 20 C57BL/6 mice underwent tendon harvest from fore- and hind limbs and endothelial cells isolation and transfection with adenoviral E4 ORF1 and red fluorescent protein labelling using a proprietary protocol. In stage 2, 158 C57BL/6 mice underwent the procedure with implantation of a 10μm fibrin glue bead (FGB) at the repair site and were randomly allocated to study group (SG) and control group (CG). Study mice received 100,000 tAECs suspended in the FGB. Control mice received the FGB only. 3 mice were sacrificed on post-operative day (POD) #3 and #7 for cell viability study. 96 mice were sacrificed at 1, 2, and 4 weeks, with 10 and 6 mice in each group and time point for biomechanical and histologic evaluations, respectively. 56 mice were sacrificed on POD 5, 10, 14, and 28 for gene expression analysis using qPCR, with 7 mice in each group and time point. Biomechanical evaluation consisted of determination of failure force and site. Histologic parameters were cell count (H&E), number of vessels (factor VIII-specific antibody), number of chondrocytes and proteoglycan content (Alcian Blue), and collagen organization (picrosirius red and polarized light microscopy). 12 evaluated genes were aggrecan (ACAN), collagen1α1 (COL1), collagen3α1 (COL3), MKX, MMP-3, 13, and 14, Runx2, scleraxis (Scx), SOX9, tenomodulin (TNM) and VEGFa.Results:Fluorescent microscopy revealed viable tAECs at the repair site on POD #3 with No fluorescent activity on POD #7. Failure force in the SG was 50% higher at 2 weeks (2.50 ± 0.55 N vs 1.86 ± 0.82 N, p = 0.012) with 65% of all failures occurring at SST mid-substance, whereas 90% of failures in the CG occurred at the repair site. There was no significant difference in failure force at 4 weeks. Cell count, number of vessels, and proteoglycan content (Figure 1) were significantly higher in the SG at all time points. The number of chondrocytes was higher in the SG at 1 week (Figure 1). Quantitative analysis showed significantly superior collagen organization in the SG (Figure 2). SCX, MKX, TNM and COL3 expression were up-regulated on POD #10 and decreased on day #28 in both groups. Expression of TNM and COL3 in the SG showed significance difference at 4 weeks. ACAN, SOX9, COL1, MMP14 expression exhibited an increase starting from POD 5, followed by a significant decline towards baseline by POD 28. In the SG, SOX9 peaked at day 14. Expression of VEGFa showed an uptrend at earlier time points (Figure 3).Conclusion:tAECs increased repair strength of SST. Histologic findings were suggestive of a more vigorous cellular and vascular response, higher proteoglycan content, and ultimately, superior collagen organization. A commensurate pattern was observed in expression of relevant genes. This approach opens a new avenue for cell-based biologic augmentation of SST repair with the goal of stimulating the intrinsic stem cell niche in tendon, as opposed to incorporating exogenous cell sources.

Pantoea agglomerans SWg2 colonizes mulberry tissues, promotes disease protection and seedling growth

Mulberry bacterial blight caused by Pseudomonas syringae pv. mori, is one of the most serious diseases on mulberry (Morus alba L.), and threatens sericulture development. An endophytic Pantoea agglomerans SWg2 (referred to as SWg2), originally isolated from roots of healthy mulberry, is extremely inhibitory activity to P. syringae. Greenhouse tests were first conducted to examine the biological control properties of SWg2. A suspension of SWg2, introduced to the host plant, significantly reduced disease symptoms caused by P. syringae. Likewise, when a suspension of SWg2 was sprayed onto plantlets 2days before inoculation with P. syringae, its disease control efficacy was up to 65.6% even after inoculation with P. syringae 18days later. Interestingly, the control effect of the traditional agrochemical streptomycin was just 33.2% at 18days post inoculation (DPI), although it was 55.6% at 9 DPI. Furthermore, SWg2 promoted the growth of mulberry seedlings. The immersion of 107CFUmL−1 suspension of SWg2 had the most distinctive growth-promoting role since the seed germination rate and radicle length development were enhanced up to 100% and 144.2%, respectively. Based on the green fluorescent protein (GFP) labeling technique and a re-isolation method, it was determined that the SWg2 strain colonized and spread in mulberry seedlings. The bacteria entered seedlings through the areas of emergence of root hairs and cracks in the rhizodermis. Eventually these cells spread to other tissues of the plant with colonization occurring mainly in the intercellular spaces. Furthermore, the GFP-tagged SWg2 strain could be detected in stems and leaves, but the quantity was smaller than that in roots. There appears to be no outward negative effects of SWg2 on treated plants and silkworms feed on them in a normal manner. This work indicates that P. agglomerans SWg2 possesses the biological potential to provide protection of its host against the pathogen of mulberry bacterial blight will at the same time promoting the growth and vigor of the host plant.

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.

SCA-1 Expression Level Identifies Quiescent Hematopoietic Stem and Progenitor Cells.

SummaryBlood cell generation depends on continuous cellular output by the sequential hierarchy of hematopoietic stem cell (HSC) and progenitor populations that all contain quiescent and actively cycling cells. Hematopoietic stem and progenitor cells (HSPCs) express the surface molecule Stem cell antigen 1 (SCA-1/LY6A). Using histone 2B-red fluorescent fusion protein label retention and cell-cycle reporter mice, we demonstrate that high SCA-1 expression (SCA-1hi) identifies not only quiescent HSCs but quiescent cells on all hierarchical levels within the lineage−SCA-1+KIT+ (LSK) population. Each transplanted SCA-1hi HSPC population also displayed self-renewal potential superior to that of the respective SCA-1lo population. SCA-1 expression is inducible by type I interferon (IFN). We show, however, that quiescence and high self-renewal capacity of cells with brighter SCA-1 expression at steady state were independent of type I IFN signaling. We conclude that SCA-1 expression levels can be used to prospectively isolate functionally heterogeneous HSPC subpopulations.

Photonic-plasmonic hybrid single-molecule nanosensor measures the effect of fluorescent labels on DNA-protein dynamics.

Current methods to study molecular interactions require labeling the subject molecules with fluorescent reporters. However, the effect of the fluorescent reporters on molecular dynamics has not been quantified because of a lack of alternative methods. We develop a hybrid photonic-plasmonic antenna-in-a-nanocavity single-molecule biosensor to study DNA-protein dynamics without using fluorescent labels. Our results indicate that the fluorescein and fluorescent protein labels decrease the interaction between a single DNA and a protein due to weakened electrostatic interaction. Although the study is performed on the DNA-XPA system, the conclusion has a general implication that the traditional fluorescent labeling methods might be misestimating the molecular interactions.

Long-lasting training in the Barnes maze prompts hippocampal spinogenesis and habituation in rats.

There is a constant need to assess spatial memory in small rodents to elucidate the basics of cognition in neuroscience experiments. Thus, the significance of the Barnes maze in the biology of hippocampal and cortical neural function cannot be overemphasized. Despite the wide use of the Barnes maze, the effect of maze task training on the structure of hippocampal neurons is yet to be elucidated. Adult Sprague-Dawley rats were subjected to intense training on the Barnes maze (3 months). Subsequently, the hippocampus (cornus ammonis and dentate gyrus) of separate sets of rats was processed for Golgi Colonnier techniques (silver impregnation) and adenoviral-green fluorescent protein labeling (immunohistochemistry). Our results showed that training the animals on the Barne maze increased spinogenesis significantly in the cornus ammonis and dentate gyrus neurons. In addition, we identified a critical time point at which the rats habituated to the trial without escaping box (the probe trial) and could not be tested further in the maze. Taken together, we deduced that a prolonged test on the dry land maze facilitated habituation and caused an increase in hippocampal dendritic spine count. As such, the dry land maze is a suitable paradigm for assessing spatial memory in rats. However, precautions should be taken in selecting suitable experimental controls on the basis of the duration of a study.

Neuronal Activation After Prolonged Immobilization: Do the Same or Different Neurons Respond to a Novel Stressor?

Despite extensive research on the impact of emotional stressors on brain function using immediate-early genes (e.g., c-fos), there are still important questions that remain unanswered such as the reason for the progressive decline of c-fos expression in response to prolonged stress and the neuronal populations activated by different stressors. This study tackles these 2 questions by evaluating c-fos expression in response to 2 different emotional stressors applied sequentially, and performing a fluorescent double labeling of c-Fos protein and c-fos mRNA on stress-related brain areas. Results were complemented with the assessment of the hypothalamic-pituitary-adrenal axis activation. We showed that the progressive decline of c-fos expression could be related to 2 differing mechanisms involving either transcriptional repression or changes in stimulatory inputs. Moreover, the neuronal populations that respond to the different stressors appear to be predominantly separated in high-level processing areas (e.g., medial prefrontal cortex). However, in low-hierarchy areas (e.g., paraventricular nucleus of the hypothalamus) neuronal populations appear to respond unspecifically. The data suggest that the distinct physiological and behavioral consequences of emotional stressors, and their implication in the development of psychopathologies, are likely to be closely associated with neuronal populations specifically activated by each stressor.

A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

Abstract WP109: Subacute Intranasal Administration of Tissue Plasminogen Activator Increases Corticospinal Tract Plasticity and Functional Recovery After Stroke in Mice

In addition to thrombolysis, tissue plasminogen activator (tPA) is involved in synaptic plasticity, dendritic remodeling and axonal outgrowth in the developing and injured CNS. We have demonstrated that tPA administered intranasally during the subacute phase after stroke enhanced neurological recovery in rats. In the present study, we examined the therapeutic benefits of intranasal tPA treatment on corticospinal tract (CST) axonal remodeling in adult mice with transgenic yellow fluorescent protein (YFP) labeling specifically in the CST subjected to permanent middle cerebral artery occlusion (MCAo). Animals were treated intranasally with 4 doses of human recombinant tPA (300 ug/dose) or saline (control) at day 7, 9, 11 and 13 after MCAo (n=10/group), respectively. An adhesive-removal test and a single-pellet reaching test were performed at 1, 3, and 7 days after MCAo, and weekly thereafter to monitor functional recovery. Animals were euthanized 4 weeks after stroke. There was no difference in lesion volume between control and tPA-treated groups (p>0.05). Compared with saline treated animals, significant functional improvements were evident in mice treated with tPA (p<0.05), as well as significantly increased CST axonal density in the denervated side of the gray matter of the cervical cord (p<0.01). We also isolated, and cultured primary embryonic YFP neurons with tPA at concentrations of 0.065, 0.65, 2.0 and 6.5 μg/ml, respectively, for 3 days, and observed that tPA dose-dependently increased neurite length and number (P<0.05). Our data suggest that delayed tPA intranasal treatment provides therapeutic benefits for neurological recovery after stroke by, at least in part, promoting CST axonal remodeling in the denervated spinal cord gray matter.

Green Fluorescent Protein Labeling of Dopaminergic Neurons in Zebrafish for the Study of Parkinson’s Diseases

The loss of dopaminergic neurons in the substantia nigra is the pathological hallmark of Parkinson s disease PD associated with the characteristic motor deficits Despite all the recent progress in the identification of genes and pathways associated with PD we still lack a complete understanding of the molecular mechanisms underpinning the disease Our goals were to develop a new transgenic zebra fish line where tyrosine hydroxylase TH positive neurons were labeled with green fluorescent protein EGFP under the control of the zebra fish tyrosine hydroxylase th promoterand to demonstrate the usefulness of the model for the study of the mechanisms underlying cell death in a toxininduced model of PD A kb gene fragment of the th promoter was isolated and cloned into a mini Tol vector followed by EGFP and microinjected to originate the Tg th EGFP zebra fish line Tg th EGFP were visualized by fluorescence microscopy for EGFP and TH expression during development The levels of EGFP mRNA were detected by RT qPCR and protein expression was examined by western blot analysis The model of neurotoxicity was generated by treating transgenic zebra fish with hydroxydopamine OHDA followed by the analysis of cell death markers and behavior EGFP expression was detected mainly in the posterior tuberculum of the ventral diencephalon vDC In the vDC TH labeled neurons of groups were correctly labeled with EGFP Further we demonstrated that these neurons in larvae and adultsare sensitive to OHDA treatment resulting in cell loss and behavioral deficits In conclusion this transgenic line constitutes a powerful tool to visualize and study dopaminergic neurons throughout the zebra fish life cycle and enables high throughput screening of molecules of interest for PD

An iminodiacetate-modified conjugated polyelectrolyte for fluorescent labeling of histidine-tagged proteins.

The iminodiacetate-Ni2+-hexahistidine system is extensively used in protein purification. In this study, an anionic conjugated polyelectrolyte (CPE) with a poly(p-phenylene ethynylene) backbone and iminodiacetate side chains, named PPEIDA, was designed and synthesized. Recognition and visualization of hexahistidine-tagged (His-tagged) proteins using PPEIDA was demonstrated.

Tetrazines in Inverse-Electron-Demand Diels–Alder Cycloadditions and Their Use in Biology

The past 10 years have seen a significant and rapid development in chemoselective reactions for labeling and controlling biological molecules in vitro but also in vivo. Here we review the class of inverse-electron-demand Diels–Alder (iEDDA) cycloadditions that have been re-discovered and developed in recent years as ideal bioorthogonal reactions and that by now play an important role in modern bioconjugation chemistry. We will especially focus on iEDDA reactions between 1,2,4,5-tetrazines and strained dienophiles and we will describe how they can be used for imaging, manipulating, and detecting biological processes. 1 Introduction 2 Inverse-Electron-Demand Diels–Alder (iEDDA) Cycloadditions 3 Synthesis of 1,2,4,5-Tetrazines 4 Synthesis of Strained Dienophiles 5 Applications in Biology 5.1 iEDDA Cycloadditions on Proteins 5.1.1 Fluorescence Labeling of Proteins via Genetic Approaches 5.1.2 Controlling Enzyme Activity in Live Cells via iEDDA Reactions 5.1.3 In vivo Imaging of Proteins via Modified Antibodies and Small Molecules 5.2 iEDDA Reactions on Nucleic Acids 5.3 iEDDA Cycloadditions To Label Glycans and Lipids 5.4 Orthogonal Bioorthogonal Reactions 5.5 Tetrazine Decaging Reactions 6 Recent Development of Other iEDDA Bioorthogonal Reactions 7 Conclusions and Outlook

Labeling proteins inside living cells using external fluorophores for microscopy.

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

RasIns: Genetically Encoded Intrabodies of Activated Ras Proteins

K- and H-Ras are the most commonly mutated genes in human tumors and are critical for conferring and maintaining the oncogenic phenotype in tumors with poor prognoses. Here, we design genetically encoded antibody-like ligands (intrabodies) that recognize active, GTP-bound K- and H-Ras. These ligands, which use the 10th domain of human fibronectin as their scaffold, are stable inside the cells and when fused with a fluorescent protein label, the constitutively active G12V mutant H-Ras. Primary selection of ligands against Ras with mRNA display resulted in an intrabody (termed RasIn1) that binds with a KD of 2.1μM to H-Ras(G12V) (GTP), excellent state selectivity, and remarkable specificity for K- and H-Ras. RasIn1 recognizes residues in the Switch I region of Ras, similar to Raf-RBD, and competes with Raf-RBD for binding. An affinity maturation selection based on RasIn1 resulted in RasIn2, which binds with a KD of 120nM and also retains excellent state selectivity. Both of these intrabodies colocalize with H-Ras, K-Ras, and G12V mutants inside the cells, providing new potential tools to monitor and modulate Ras-mediated signaling. Finally, RasIn1 and Rasin2 both display selectivity for the G12V mutants as compared with wild-type Ras providing a potential route for mutant selective recognition of Ras.

Green fluorescent protein labeling of food pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis

Labeling of bacteria with marker genes, such as green fluorescent protein, is a useful and practicable tool for tracking and enumerating bacterial cells in a complex environment e.g. discrimination from the indigenous background population. In this study, novel TurboGFP prokaryotic expression vector was utilized for labeling of Yersinia species. Y. enterocolitica biovar 1A, biovar 2, biovar 4 and Y. pseudotuberculosis were successfully transformed with the vector and expressed bright green fluorescence that was even detectable visually by eye. No adverse effects were observed in growth behavior of the labeled strains compared to wild type (parental) strains and vector maintenance for longer time periods could be achieved for Y. enterocolitica biovar 1A, Y. enterocolitica biovar 2 and Y. pseudotuberculosis.

Genetic code expansion for multiprotein complex engineering

We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure-function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies.