Fluorescent Protein mCherry Research Articles

Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12

BackgroundIn bacteria, many transcription activator and repressor proteins regulate multiple transcription units that are often distally distributed on the bacterial genome. To investigate the subcellular location of DNA bound proteins in the folded bacterial nucleoid, fluorescent reporters have been developed which can be targeted to specific DNA operator sites. Such Fluorescent Reporter-Operator System (FROS) probes consist of a fluorescent protein fused to a DNA binding protein, which binds to an array of DNA operator sites located within the genome. Here we have developed a new FROS probe using the Escherichia coli MalI transcription factor, fused to mCherry fluorescent protein. We have used this in combination with a LacI repressor::GFP protein based FROS probe to assess the cellular location of commonly regulated transcription units that are distal on the Escherichia coli genome.ResultsWe developed a new DNA binding fluorescent reporter, consisting of the Escherichia coli MalI protein fused to the mCherry fluorescent protein. This was used in combination with a Lac repressor:green fluorescent protein fusion to examine the spatial positioning and possible co-localisation of target genes, regulated by the Escherichia coli AraC protein. We report that induction of gene expression with arabinose does not result in co-localisation of AraC-regulated transcription units. However, measurable repositioning was observed when gene expression was induced at the AraC-regulated promoter controlling expression of the araFGH genes, located close to the DNA replication terminus on the chromosome. Moreover, in dividing cells, arabinose-induced expression at the araFGH locus enhanced chromosome segregation after replication.ConclusionRegions of the chromosome regulated by AraC do not colocalise, but transcription events can induce movement of chromosome loci in bacteria and our observations suggest a role for gene expression in chromosome segregation.

Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis.

An extracellular layer of exopolysaccharides (EPS) covers the surface of some Bifidobacterium animalis subsp. lactis strains, which could be of relevance for its probiotic performance. In order to understand the functional characteristics of B. animalis subsp. lactis, two isogenic strains that differ in their EPS-producing phenotype, due to a single mutation in the gene Balat_1410, were studied. By means of a double crossover recombination strategy, successfully used for the first time in bifidobacteria, Balat_1410 in the type strain B. animalis subsp. lactis DSM10140 was replaced by a mutated gene containing a non-synonymous mutation previously associated with the appearance of a mucoid-ropy phenotype. Nuclear magnetic resonance and SEC-MALS analyses showed that the novel strain harboring the mutation acquired a ropy phenotype, due to the production of a high molecular weight (HMW)-EPS that is not produced in the wild-type strain. Fluorescence labeling of both strains with two fluorescent proteins, m-Cherry and Green Fluorescent Protein, was achieved by expressing the corresponding genes under the control of a native selected promoter (the elongation factor Tu promoter). Remarkably, qualitative and quantitative fluorescence analyses demonstrated that the ropy strain displays a lower capability to adhere to human intestinal epithelial cells. In addition, the presence of the HMW-EPS reduced the capability of the producing strain to form biofilms upon three different abiotic surfaces. This work also highlights the fact that different EPS confer variable functional characteristics to the bifidobacterial surface, which may be relevant for the performance of B. animalis subsp. lactis as a probiotic. The construction of molecular tools allowing the functional characterization of surface structures in next generation probiotics is still a challenging issue that deserves further attention, given the relevant role that such molecules must play in the interaction with the host.

Projection targets of medial frontal D1DR-expressing neurons.

Prefrontal neurons expressing D1-type dopamine receptors (D1DRs) have been implicated in a variety of cognitive processes including working memory and timing. Although D1DRs are most strongly expressed on layer V/VI projection neurons, it is unknown which brain areas are specifically targeted by these projections. Here we selectively marked D1DR neurons using cre-loxP techniques with AAV carrying mCherry fluorescent protein, and traced projection targets of D1DR+ neurons in the mouse medial frontal cortex (MFC). We found relatively strong MFC D1DR+ projections to cortical areas as well as projections to basal ganglia and thalamic nuclei. We found relatively weaker MFC D1DR+ projections to the brainstem, hypothalamus, and other subcortical nuclei. These data intimate that MFC D1DR+ projections are well-positioned to powerfully influence cortical processing and have subcortical specificity. Thus MFC D1DR+ projection neurons may play a key role in tuning cortical networks during goal-directed behavior.

Eimeria tenella protein trafficking: differential regulation of secretion versus surface tethering during the life cycle

Eimeria spp. are intracellular parasites that have a major impact on poultry. Effective live vaccines are available and the development of reverse genetic technologies has raised the prospect of using Eimeria spp. as recombinant vectors to express additional immunoprotective antigens. To study the ability of Eimeria to secrete foreign antigens or display them on the surface of the sporozoite, transiently transfected populations of E. tenella expressing the fluorescent protein mCherry, linked to endogenous signal peptide (SP) and glycophosphatidylinositol-anchor (GPI) sequences, were examined. The SP from microneme protein EtMIC2 (SP2) allowed efficient trafficking of mCherry to cytoplasmic vesicles and following the C-terminal addition of a GPI-anchor (from surface antigen EtSAG1) mCherry was expressed on the sporozoite surface. In stable transgenic populations, mCherry fused to SP2 was secreted into the sporocyst cavity of the oocysts and after excystation, secretion was detected in culture supernatants but not into the parasitophorous vacuole after invasion. When the GPI was incorporated, mCherry was observed on the sporozites surface and in the supernatant of invading sporozoites. The proven secretion and surface exposure of mCherry suggests that antigen fusions with SP2 and GPI of EtSAG1 may be promising candidates to examine induction of protective immunity against heterologous pathogens.

Abstract 967: Development of fluorescent-cell based high throughput assay to identify novel therapeutics of bladder cancer cells through upregulation of SPARC expression

Abstract Bladder cancer is the most common malignancy affecting the urinary system with an estimated 76,960 new cases and projected 16,390 deaths in 2016 in the United States. The role of SPARC in various cancers is contextual and differs based on the tumor classification, malignancy and progression. We have recently reported the tumor suppressor effect of SPARC in urinary bladder cancer. We have shown that the loss of SPARC in bladder cancer resulted accelerated bladder carcinogenesis and metastasis multiple pathways involving cell cycle deregulation, inflammation and angiogenesis. We have also reported that SPARC protein expression significantly decreased in the cancerous compartment in advanced human bladder cancer as well as in carcinogen-induced murine urothelial cancer. However, the mechanism of the downregulation of SPARC expression in cancer in general and in bladder cancer in particular is still unraveled. The purpose of our study is to develop a robust, high throughput assay to identify therapeutic candidates as regulators of SPARC expression in bladder cancer. We cloned SPARC promoter in a third generation lentiviral vector coupled with fluorescent GFP or mCherry fluorescent protein to quantitatively measure SPARC expression. We generated stable bladder cancer cell lines expressing SPARC expression in bladder cancer cell lines. UMUC3 bladder cancer cells were transduced with lentiviral vector to generate SPARC-promoter reporter system to screen regulators of SPARC expression. Reporter cells were plated in 96 well plates and treated with 10uM of FDA-approved and natural product drug libraries. Real time monitoring of changes in SPARC expression was done by measuring GFP fluorescence intensity using IncuCyte® ZOOM Live Imaging Analysis System. We identified drugs that consistently exhibited dosage-dependent augmentation or down-regulation of SPARC expression in UMUC3 cells. Our screens identified novel up-regulators of SPARC expression that are both standard of care and novel bladder cancer therapeutics. Our efforts produced an efficient robust assay to identify novel therapeutics and regulators of the tumor suppressor SPARC in the bladder cancer ecosystem that can be used in the adjuvant and neoadjuvant settings. Citation Format: Chirayu M. Patel. Development of fluorescent-cell based high throughput assay to identify novel therapeutics of bladder cancer cells through upregulation of SPARC expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 967. doi:10.1158/1538-7445.AM2017-967

Comparative transcriptomic analysis identifies evolutionarily conserved gene products in the vertebrate renal distal convoluted tubule.

Understanding the molecular basis of the complex regulatory networks controlling renal ion transports is of major physiological and clinical importance. In this study, we aimed to identify evolutionarily conserved critical players in the function of the renal distal convoluted tubule (DCT) by a comparative transcriptomic approach. We generated a transgenic zebrafish line with expression of the red fluorescent mCherry protein under the control of the zebrafish DCT-specific promoter of the thiazide-sensitive NaCl cotransporter (NCC). The mCherry expression was then used to isolate from the zebrafish mesonephric kidneys the distal late (DL) segments, the equivalent of the mammalian DCT, for subsequent RNA-seq analysis. We next compared this zebrafish DL transcriptome to the previously established mouse DCT transcriptome and identified a subset of gene products significantly enriched in both the teleost DL and the mammalian DCT, including SLCs and nuclear transcription factors. Surprisingly, several of the previously described regulators of NCC (e.g., SPAK, KLHL3, ppp1r1a) in the mouse were not found enriched in the zebrafish DL. Nevertheless, the zebrafish DL expressed enriched levels of related homologues. Functional knockdown of one of these genes, ppp1r1b, reduced the phosphorylation of NCC in the zebrafish pronephros, similar to what was seen previously in knockout mice for its homologue, Ppp1r1a. The present work is the first report on global gene expression profiling in a specific nephron portion of the zebrafish kidney, an increasingly used model system for kidney research. Our study suggests that comparative analysis of gene expression between phylogenetically distant species may be an effective approach to identify novel regulators of renal function.

Efficient switching of mCherry fluorescence using chemical caging

Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantum-mechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores.

The development of fluorescent protein tracing vectors for multicolor imaging of clinically isolated Staphylococcus aureus

Recent advances in fluorescent protein technology provide a wide variety of biological imaging applications; however current tools for bio-imaging in the Gram-positive bacterium Staphylococcus aureus has necessitated further developments for fluorescence intensity and for a multicolor palette of fluorescent proteins. To enhance the expression of multicolor fluorescent proteins in clinical S. aureus strains, we developed new fluorescent protein expression vectors, containing the blaZ/sodp promoter consisting of the β-lactamase gene (blaZ) promoter and the ribosome binding site (RBS) of superoxide dismutase gene (sod). We found S. aureus-adapted GFP (GFPsa) driven by the blaZ/sodp promoter was highly expressed in the S. aureus laboratory strain RN4220, but not in the clinical strains, MW2 and N315, harboring the endogenous blaI gene, a repressor of the blaZ gene promoter. We therefore constructed a constitutively induced blaZ/sodp promoter (blaZ/sodp(Con)) by introducing substitution mutations into the BlaI binding motif, and this modification allowed enhanced expression of the multicolor GFP variants (GFPsa, EGFP, mEmerald, Citrine, Cerulean, and BFP) as well as codon-optimized reef coral fluorescent proteins (mCherry and AmCyan) in the S. aureus clinical strains. These new fluorescent probes provide new tools to enhance expression of multicolor fluorescent proteins and facilitate clear visualization of clinical S. aureus strains.

Coupling optogenetics and light-sheet microscopy to study signal transduction in vivo

Optogenetics allows precise, fast and reversible intervention in biological processes. Coupled with light-sheet microscopy, this approach allows unparalleled insight into the regulation of signaling pathways and cellular processes from a spatial and temporal perspective in vivo. To develop this method, we investigated the spatial and temporal regulation of canonical Wnt signaling during anterior-posterior patterning of the Drosophila embryonic epidermis. Cryptochrome C (Cry2) from Arabidopsis Thaliana was fused to mCherry fluorescent protein and Drosophila β-catenin to form an easy to visualize optogenetic switch. Blue light illumination caused reversible oligomerization of the fusion protein and inhibited downstream Wnt signaling in vitro and in vivo. Temporal inactivation of β-catenin showed that Wnt signaling is required not only for Drosophila patterning formation, but also for maintenance throughout development. By applying light-sheet microscopy to activate the pathway and then record subsequent behavior in toto, we show precise spatial regulation of Wnt signaling in vivo. We anticipate that this method will be easily extendable to other developmental signaling pathways and many other experimental systems.

Approaches to Controlled Co-Amplification of Genes for Production of Biopharmaceuticals: Study of the Insertion and Amplification Dynamics of Genetic Cassettes in the Genome of Chinese Hamster Ovary Cells during Co-Expression of Compatible Pair of Plasmids.

Plasmid vector family p1.1 based on non-coding regions of Chinese hamster housekeeping gene EEF1A and concatemer of Epstein-Barr virus terminal repeat increases the frequency of genome integration and provides rapid amplification of the target genes in the genome. For a pair of fluorescent proteins eGFP and mCherry it was shown that p1.1 vectors bearing dihydrofolate reductase and glutamine synthetase selection markers upon co-transfection into CHO DG44 cell line allow obtaining a polyclonal cell population in which ~70% of cells express both genes. The subsequent one-step gene amplification of the genome-integrated genetic cassettes under the selective pressure of increased concentrations of methotrexate can increase the expression of both integrated genes up to 8.2% eGFP and 9.9% mCherry of total protein. This approach can be used for the development of cell lines for the production of functional heterodimeric proteins, e.g. polypeptide hormones and therapeutic antibodies.

Checks and Balances-The Limits of β-Cell Endurance to ER Stress.

As James Madison remarked, “the great question now to be decided … is, whether checks and balances sufficient for the purposes of order, justice, and the general good, may not be created by a proper division and distribution of power among different bodies, differently constituted, but all deriving their existence from the elective principle, and bound by a responsible tenure of their trusts” (1). But which are the checks and balances that allow pancreatic β-cells to preserve their most differentiated function, i.e., the synthesis and release of insulin, and what are the limits of endurance of these cells when facing severe stress? To evaluate this, Pappalardo et al. (2) made ingenious use of small interfering RNAs (siRNAs). RNA interference (RNAi) is triggered by endogenously produced or exogenously introduced RNA pairs that have complementarity with endogenous mRNAs, leading to their degradation and consequent “silencing” of the corresponding gene and its encoded protein. This provides the possibility of silencing individual genes in selected cells to understand their function(s). Instead of targeting individual genes, however, Pappalardo et al. (2) performed a whole-genome RNAi screen (targeting in parallel 22,000 mouse genes) to identify the genes that, when inhibited, would affect insulin transcription. This approach is based on the use of a mouse insulin-producing cell line, MIN6, engineered to express both a copy of the human insulin promoter driving a fluorescent EGFP reporter and the mCherry fluorescent protein under the control of a constitutively active viral promoter as an internal control. When one of the siRNAs introduced in the cells inhibits a positive regulator of the insulin promoter, …

An Experimental and Molecular Dynamics Study of Red Fluorescent Protein mCherry in Novel Aqueous Amino Acid Ionic Liquids.

The search for biocompatible ionic liquids (ILs) with novel biochemical and biomedical applications has recently gained greater attention. In this report, we characterize the effects of two novel amino acid-based aqueous ILs composed of tetramethylguanidinium (TMG) and amino acids on the structure and stability of a widely used red fluorescent protein (mCherry). Our experimental data shows that while the aspartic acid-based IL (TMGAsp) has effects similar to previously studied conventional ILs (BMIBF4, EMIAc, and TMGAc), the alanine-based IL (TMGAla) has a much stronger destabilization effect on the protein structure. Addition of 0.30 M TMGAla to mCherry decreases the unfolding temperature from 83 to 60 °C. Even at 25 °C, TMGAla results in a blue shift of the mCherry absorbance and fluorescence peaks and an increased Stokes shift. Molecular dynamics simulations show that the chromophore conformation and its interaction with mCherry with TMGAla are changed relative to those with TMGAsp or in the absence of ILs. Protein-ILs contact analysis indicates that the mCherry-Asp interactions are hydrophilic but the (fewer) mCherry-Ala interactions are more hydrophobic and may modulate the TMG interaction with the protein. Hence, the anion hydrophobicity may explain the special TMGAla destabilization of mCherry.

Bio-orthogonal conjugation and enzymatically triggered release of proteins within multi-layered hydrogels.

Controlling protein presentation within biomaterials is important for modulating interactions with biological systems. For example, native tissues are composed of subunits with different matrix compositions (proteins, stiffness) that dynamically interact with cells, influencing function and fate. Toward mimicking such temporally-regulated and spatially-defined microenvironments, we utilize bio-orthogonal click chemistry and protein engineering to create hydrogels with distinct regions of proteins and modify them over time. Through nonsense codon replacement, we site-specifically functionalize large proteins with i) azides for covalent conjugation and ii) an enzymatic cleavage site for user-defined release from hydrogels. Our results exemplify not only the ability to create unique bio-functionalized hydrogels with controlled mechanical properties, but also the potential for creating interesting interfaces for cell culture and tissue engineering applications.

MP39-19 SUNITINIB DRIVES CELL-TO-CELL MITOCHONDRIAL TRAFFICKING IN KIDNEY CANCER

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I1 Apr 2017MP39-19 SUNITINIB DRIVES CELL-TO-CELL MITOCHONDRIAL TRAFFICKING IN KIDNEY CANCER Jason Scovell, Juan Hernandez, and Richard Link Jason ScovellJason Scovell More articles by this author , Juan HernandezJuan Hernandez More articles by this author , and Richard LinkRichard Link More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1193AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES When grown under stressed conditions, a subset of neoplastic cells can undergo cell-to-cell trafficking of mitochondria through dynamic structures termed tunneling nanotubes. This horizontal mitochondrial transfer (HMT) is a relatively newly described phenomenon that may contribute to chemotherapeutic resistance in several non-urologic cancers. Since HMT might represent a novel mechanism of acquired chemoresistance in renal cell carcinoma (RCC), we sought to determine if HMT occurs in response to treatment with Sunitinib, a first line chemotherapeutic agent. METHODS RCC cell lines (A498 and 786-O) were differentially labeled by lentiviral transduction of cytoplasm-localizing enhanced green fluorescent protein (EGFP) or mitochondria-localizing mCherry. Mitochondrial localization of mCherry (mito-mCherry) was achieved by tagging the fluorescent protein with the cyclooxygenase-8 mitochondria targeting sequence. Differentially labeled RCC cells were cultured at a 1:1 ratio and were treated with sunitinib (0uM, 1.25uM, 2.5um, 5uM, 10uM, and 20uM) for 24 hours. HMT was quantified by flow cytometry and visualized by deconvolution microscopy. RESULTS HMT occurred between differentially labeled RCC cells. Co-culture of mito-mCherry A498 cells with EGFP+ A498, 786-O, and ACHN cells demonstrated mitochondrial transfer at all concentrations of sunitinib tested. 2.4% of EGFP+ A498 cells received mCherry+ mitochondria in co-culture at 0uM of sunitinib. Mitochondrial transfer was enhanced by sunitinib treatment at all doses tested (e.g. 4.26% at 10uM sunitinib). This phenomenon was not cell line specific as EGFP labeled 786-O cells also acquired mCherry+ mitochondria (8.3%). Deconvolution microscopy of fixed co-cultured cells demonstrated mCherry+ mitochondria within EGFP+ 786-O cells. CONCLUSIONS Here we demonstrate for the first time that sunitinib exposure potentiates cell-to-cell transfer of mitochondria in kidney cancer cell lines. The biologic significance of this process in vivo remains to be determined. Our hypothesis is that HMT may contribute to clinically relevant acquired chemoresistance in RCC and experiments are under way to explore this possibility further. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e501 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Jason Scovell More articles by this author Juan Hernandez More articles by this author Richard Link More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells

The periphery of epithelial cells is shaped by opposing cytoskeletal physical forces generated predominately by two dynamic force generating systems—growing microtubule ends push against the boundary from the cell center, and the actin cortex contracts the attached plasma membrane. Here we investigate how changes to the structure and dynamics of the actin cortex alter the dynamics of microtubules. Current drugs target actin polymerization and contraction to reduce cell division and invasiveness; however, the impacts on microtubule dynamics remain incompletely understood. Using human MCF-7 breast tumor cells expressing GFP-tagged microtubule end-binding-protein-1 (EB1) and coexpression of cytoplasmic fluorescent protein mCherry, we map the trajectories of growing microtubule ends and cytoplasmic boundary respectively. Based on EB1 tracks and cytoplasmic boundary outlines, we calculate the speed, distance from cytoplasmic boundary, and straightness of microtubule growth. Actin depolymerization with Latrunculin-A reduces EB1 growth speed as well as allows the trajectories to extend beyond the cytoplasmic boundary. Blebbistatin, a direct myosin-II inhibitor, reduced EB1 speed and yielded less straight EB1 trajectories. Inhibiting signaling upstream of myosin-II contractility via the Rho-kinase inhibitor, Y-27632, altered EB1 dynamics differently from Blebbistatin. These results indicate that reduced actin cortex integrity can induce distinct alterations in microtubule dynamics. Given recent findings that tumor stem cell characteristics are increased by drugs which reduce actin contractility or stabilize microtubules, it remains important to clearly define how cytoskeletal drugs alter the interactions between these two filament systems in tumor cells.

An Engineered Bacterial Display System for the Development of a High‐Throughput Screen for Compounds that Block Amyloid Fiber Formation

The molecular basis for a number of neuronal diseases, such as Alzheimer's and Parkinson's, is aberrant peptide or protein aggregation that result in the formation of amyloid fibers. For example, the Aβ-42 peptide is a molecular component of the plaques found in the brains of Alzheimer's patients, and the protein α-synuclein is a constituent of Lewy bodies, a hallmark of Parkinson's disease. The exact role these proteins play in disease is currently not completely clear but it is generally believed that aberrant self-association contributes in large part to the disease state. For Aβ-42 it is not currently known whether it is low-order complexes (e.g., trimers, tetramers, etc.) or higher-order aggregates that lead to cell death and neuronal degradation. With Parkinson's disease, genetic variations in the gene for α-synuclein give rise to overexpression or aggregate prone variants of the protein. Regardless of the exact disease mechanism, or what level of self-association causes disease, it is generally recognized that blocking the aberrant self-association of these protein-based elements is of high scientific merit. To this end we have engineered a fluorescence-based Bacterial Surface Display (BSD) system that enables a relatively high expression level of a protein-of-interest (POI) on the outer surface of E. coli. Upon expression the POI is fused to an N-terminal periplasmic leader sequence, and to its C-terminus the red fluorescent protein mCherry, and finally a transmembrane protein that functions to anchor the entire fusion construct in the bacterial outer membrane. When the BSD system is used to display Aβ-42, or α-synuclein, the bacterial cells are driven to strongly self-associate. Thus far we have successfully demonstrated the utility of the system using antibodies against Aβ-42. Drug-like compounds that have been proven to block amyloid fiber formation in vitro will also be tested using the Aβ-42 and α-synuclein BSD systems. Immediate and longer-range goals entail the development of a high-throughput screen for additional compounds that bind Aβ-42 and α-synuclein and block their ability to self-associate into amyloid fibers. In addition, the BSD system will also be used for the directed evolution of a small, stable test protein that will be engineered to bind immobilized Aβ-42 and α-synuclein. The test protein, the β1 domain of streptococcal protein-G (Gβ1), consists of an α-helix overlaying a β-sheet made up of two β-hairpins. Codons for select residue positions, located at the edge of one side of the β-sheet, will be randomized and the resulting library displayed using the BSD system. The bacteria displaying the library will be incubated against immobilized Aβ-42 and α-synuclein where successful complex formation (binding) will function as the selection process. The β-hairpin, which contains the amino acids that drive binding, will be expressed/produced separately and assessed for its ability to bind the Aβ-42 and α-synuclein and block their ability to form amyloid fibers. A long-term goal is to convert this β-hairpin peptide into a more drug-like molecule using chemical biology methods such as converting the α amino acids into β forms and using unnatural D stereoisomers of amino acids. The ultimate goal is to develop drugs that effectively combat the devastating neuronal diseases caused by aberrant protein aggregation. Support or Funding InformationThis material is based upon work supported in part by the U.S. Army Research Laboratory and the U.S. Army Research Office under grant number W911NF-13-1-0155. Bacterial Surface Display of Aggregation-Prone Proteins. (A) Aβ-42 peptide (B) α-synuclein

A Microplate Reader-Based System for Visualizing Transcriptional Activity During in vivo Microbial Interactions in Space and Time

Here, we report the development of a microplate reader-based system for visualizing gene expression dynamics in living bacterial cells in response to a fungus in space and real-time. A bacterium expressing the red fluorescent protein mCherry fused to the promoter region of a regulator gene nunF indicating activation of an antifungal secondary metabolite gene cluster was used as a reporter system. Time-lapse image recordings of the reporter red signal and a green signal from fluorescent metabolites combined with microbial growth measurements showed that nunF-regulated gene transcription is switched on when the bacterium enters the deceleration growth phase and upon physical encounter with fungal hyphae. This novel technique enables real-time live imaging of samples by time-series multi-channel automatic recordings using a microplate reader as both an incubator and image recorder of general use to researchers. The technique can aid in deciding when to destructively sample for other methods e.g. transcriptomics and mass spectrometry imaging to study gene expression and metabolites exchanged during the interaction.

Development of Lactococcus lactis encoding fluorescent proteins, GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.

The GCaMP-R Family of Genetically Encoded Ratiometric Calcium Indicators.

We report on GCaMP-Rs, a new family of genetically encoded ratiometric calcium indicators that extend the virtues of the GCaMP proteins to ratiometric measurements. We have engineered a tandem construct of calcium-dependent GCaMP and calcium-independent mCherry fluorescent proteins. The tandem design assures that the two proteins localize in the same cellular compartment(s) and facilitates pixelwise ratiometric measurements; however, Förster resonance energy transfer (FRET) between the fluorophores reduces brightness of the sensor by up to half (depending on the GCaMP variant). To eliminate FRET, we introduced a rigid α-helix, the ER/K helix, between GCaMP and mCherry. Avoiding FRET significantly increases the brightness (notably, even at low calcium concentrations), the signal-to-noise ratio, and the dynamic range.

Self-Assembled Gold Nanoparticle-Fluorescent Protein Conjugates as Platforms for Sensing Thiolate Compounds via Modulation of Energy Transfer Quenching.

The ability of Au and other metal nanostructures to strongly quench the fluorescence of proximal fluorophores (dyes and fluorescent proteins) has made AuNP conjugates attractive for use as platforms for sensor development based on energy transfer interactions. In this study, we first characterize the energy transfer quenching of mCherry fluorescent proteins immobilized on AuNPs via metal-histidine coordination, where parameters such as NP size and number of attached proteins are varied. Using steady-state and time-resolved fluorescence measurements, we recorded very high mCherry quenching, with efficiency reaching ∼95-97%, independent of the NP size or number of bound fluorophores (i.e., conjugate valence). We further exploited these findings to develop a solution phase sensing platform targeting thiolate compounds. Energy transfer (ET) was employed as a transduction mechanism to monitor the competitive displacement of mCherry from the Au surface upon the introduction of varying amounts of thiolates with different size and coordination numbers. Our results show that the competitive displacement of mCherry depends on the thiolate concentration, time of reaction, and type of thiol derivatives used. Further analysis of the PL recovery data provides a measure for the equilibrium dissociation constant (Kd-1) for these compounds. These findings combined indicate that the AuNP-fluorescent protein conjugates may offer a potentially useful platform for thiol sensing both in solution and in cell cultures.