scholarly journals A fluorescent protein-readout for transcriptional activity reveals regulation of APP nuclear signaling by phosphorylation sites

2019 ◽  
Vol 400 (9) ◽  
pp. 1191-1203 ◽  
Author(s):  
Uwe Konietzko ◽  
Manuel T. Gersbacher ◽  
Jeremy Streuli ◽  
Maik Krüger ◽  
Sarina Thöni ◽  
...  
Keyword(s):  
Single Cell ◽  
Transcriptional Activity ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Screening Methods ◽  
Single Cell Level ◽  
Cell Level ◽  
Transcription System ◽  
Nuclear Signaling ◽  
Highly Sensitive

Abstract Signaling pathways that originate at the plasma membrane, including regulated intramembrane proteolysis (RIP), enable extracellular cues to control transcription. We modified the yeast Gal4 transcription system to study the nuclear translocation of transcriptionally active complexes using the fluorescent protein citrine (Cit) as a reporter. This enabled highly sensitive quantitative analysis of transcription in situ at the single cell level. The Gal4/UAS-Cit transcription assay displayed a sigmoidal response limited by the number of integrated reporter cassettes. We validated the assay by analyzing nuclear translocation of the amyloid precursor protein (APP) intracellular domain (AICD) and confirmed the requirement of Fe65 for nuclear translocation of AICD. In addition to the strong on-off effects on transcriptional activity, the results of this assay establish that phosphorylation modifies nuclear signaling. The Y682F mutation in APP showed the strongest increase in Cit expression, underscoring its role in regulating Fe65 binding. Together, we established a highly sensitive fluorescent protein-based assay that can monitor transcriptional activity at the single cell level and demonstrate that AICD phosphorylation affects Fe65 nuclear activity. This assay also introduces a platform for future single cell-based drug screening methods for nuclear translocation.

scholarly journals NADH Fluorescence Lifetime Imaging Microscopy Reveals Selective Mitochondrial Dysfunction in Neurons Overexpressing Alzheimer’s Disease–Related Proteins

2021 ◽  
Vol 8 ◽  
Author(s):  
Moritz A. Niederschweiberer ◽  
Patrick M. Schaefer ◽  
Larry N. Singh ◽  
Ludwig Lausser ◽  
Devyani Bhosale ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Single Cell ◽  
Mitochondrial Function ◽  
Primary Neurons ◽  
Single Cell Level ◽  
Cell Level ◽  
Lifetime Imaging ◽  
Highly Sensitive ◽  
Related Proteins ◽  
Subcellular Level

Alzheimer’s disease (AD), the most prevalent form of dementia, affects globally more than 30 million people suffering from cognitive deficits and neuropsychiatric symptoms. Substantial evidence for the involvement of mitochondrial dysfunction in the development and/or progression of AD has been shown in addition to the pathological hallmarks amyloid beta (Aβ) and tau. Still, the selective vulnerability and associated selective mitochondrial dysfunction cannot even be resolved to date. We aimed at optically quantifying mitochondrial function on a single-cell level in primary hippocampal neuron models of AD, unraveling differential involvement of cell and mitochondrial populations in amyloid precursor protein (APP)-associated mitochondrial dysfunction. NADH lifetime imaging is a highly sensitive marker-free method with high spatial resolution. However, deciphering cellular bioenergetics of complex cells like primary neurons has still not succeeded yet. To achieve this, we combined highly sensitive NADH lifetime imaging with respiratory inhibitor treatment, allowing characterization of mitochondrial function down to even the subcellular level in primary neurons. Measuring NADH lifetime of the same neuron before and after respiratory treatment reveals the metabolic delta, which can be taken as a surrogate for cellular redox capacity. Correlating NADH lifetime delta with overexpression strength of Aβ-related proteins on the single-cell level, we could verify the important role of intracellular Aβ-mediated mitochondrial toxicity. Subcellularly, we could demonstrate a higher respiration in neuronal somata in general than dendrites, but a similar impairment of somatic and dendritic mitochondria in our AD models. This illustrates the power of NADH lifetime imaging in revealing mitochondrial function on a single and even subcellular level and its potential to shed light into bioenergetic alterations in neuropsychiatric diseases and beyond.

High resolution FTIR imaging provides automated discrimination and detection of single malaria parasite infected erythrocytes on glass

2016 ◽  
Vol 187 ◽  
pp. 341-352 ◽  
Author(s):  
David Perez-Guaita ◽  
Dean Andrew ◽  
Philip Heraud ◽  
James Beeson ◽  
David Anderson ◽  
...  
Keyword(s):  
Single Cell ◽  
Numerical Aperture ◽  
Single Cell Level ◽  
Cell Level ◽  
High Quality ◽  
Pixel Resolution ◽  
Ftir Microscopy ◽  
Synchrotron Source ◽  
Average Spectrum ◽  
Highly Sensitive

New highly sensitive tools for malaria diagnostics are urgently needed to enable the detection of infection in asymptomatic carriers and patients with low parasitemia. In pursuit of a highly sensitive diagnostic tool that can identify parasite infections at the single cell level, we have been exploring Fourier transform infrared (FTIR) microscopy using a Focal Plane Array (FPA) imaging detector. Here we report for the first time the application of a new optic configuration developed by Agilent that incorporates 25× condenser and objective Cassegrain optics with a high numerical aperture (NA = 0.81) along with additional high magnification optics within the microscope to provide 0.66 micron pixel resolution (total IR system magnification of 61×) to diagnose malaria parasites at the single cell level on a conventional glass microscope slide. The high quality images clearly resolve the parasite's digestive vacuole demonstrating sub-cellular resolution using this approach. Moreover, we have developed an algorithm that first detects the cells in the infrared image, and secondly extracts the average spectrum. The average spectrum is then run through a model based on Partial Least Squares-Discriminant Analysis (PLS-DA), which diagnoses unequivocally the infected from normal cells. The high quality images, and the fact this measurement can be achieved without a synchrotron source on a conventional glass slide, shows promise as a potential gold standard for malaria detection at the single cell level.

scholarly journals Generation of genetic tools for gauging multiple gene expression at single cell level

2021 ◽  
Author(s):  
Marta Mellini ◽  
Massimiliano Lucidi ◽  
Francesco Imperi ◽  
Paolo Visca ◽  
Livia Leoni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Image Processing ◽  
Single Cell ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Phenotypic Heterogeneity ◽  
Single Cell Level ◽  
Cell Level ◽  
Multiple Gene ◽  
Genetic Tools

Key microbial processes in many bacterial species are heterogeneously expressed in single cells of bacterial populations. However, the paucity of adequate molecular tools for live, real-time monitoring of multiple gene expression at the single cell level has limited the understanding of phenotypic heterogeneity. In order to investigate phenotypic heterogeneity in the ubiquitous opportunistic pathogen Pseudomonas aeruginosa, a genetic tool that allows gauging multiple gene expression at the single cell level has been generated. This tool, named pRGC, consists in a promoter-probe vector for transcriptional fusions that carries three reporter genes coding for the fluorescent proteins mCherry, green fluorescent protein (GFP) and cyan fluorescent protein (CFP). The pRGC vector has been characterized and validated via single cell gene expression analysis of both constitutive and iron-regulated promoters, showing clear discrimination of the three fluorescence signals in single cells of a P. aeruginosa population, without the need of image-processing for spectral crosstalk correction. In addition, two pRGC variants have been generated for either i) integration of the reporter gene cassette into a single neutral site of P. aeruginosa chromosome, that is suitable for long-term experiments in the absence of antibiotic selection, or ii) replication in bacterial genera other than Pseudomonas. The easy-to-use genetic tools generated in this study will allow rapid and cost-effective investigation of multiple gene expression in populations of environmental and pathogenic bacteria, hopefully advancing the understanding of microbial phenotypic heterogeneity. IMPORTANCE Within a bacterial population single cells can differently express some genes, even though they are genetically identical and experience the same chemical and physical stimuli. This phenomenon, known as phenotypic heterogeneity, is mainly driven by gene expression noise and results in the emergence of bacterial sub-populations with distinct phenotypes. The analysis of gene expression at the single cell level has shown that phenotypic heterogeneity is associated with key bacterial processes, including competence, sporulation and persistence. In this study, new genetic tools have been generated that allow easy cloning of up to three promoters upstream of distinct fluorescent genes, making it possible to gauge multiple gene expression at the single cell level by fluorescent microscopy, without the need of advanced image-processing procedures. A proof of concept has been provided by investigating iron-uptake and iron-storage gene expression in response to iron availability in P. aeruginosa.

scholarly journals Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level

2000 ◽  
Vol 97 (13) ◽  
pp. 7260-7265 ◽  
Author(s):  
V. Baubet ◽  
H. Le Mouellic ◽  
A. K. Campbell ◽  
E. Lucas-Meunier ◽  
P. Fossier ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Single Cell ◽  
Fluorescent Protein ◽  
Single Cell Level ◽  
Cell Level ◽  
Green Fluorescent

Fluorescent protein-based FRET sensor for intracellular monitoring of redox status in bacteria at single cell level

2014 ◽  
Vol 406 (28) ◽  
pp. 7195-7204 ◽  
Author(s):  
Bobin George Abraham ◽  
Ville Santala ◽  
Nikolai V. Tkachenko ◽  
Matti Karp
Keyword(s):  
Single Cell ◽  
Fluorescent Protein ◽  
Redox Status ◽  
Single Cell Level ◽  
Cell Level

scholarly journals Bacterial Activity in the Rhizosphere Analyzed at the Single-Cell Level by Monitoring Ribosome Contents and Synthesis Rates

2000 ◽  
Vol 66 (2) ◽  
pp. 801-809 ◽  
Author(s):  
Cayo Ramos ◽  
Lars Mølbak ◽  
Søren Molin
Keyword(s):  
Growth Rate ◽  
Single Cell ◽  
Monitoring System ◽  
Fluorescent Protein ◽  
Transient Gene Expression ◽  
Single Cell Level ◽  
Cell Level ◽  
Gene Encoding ◽  
Rate Dependent ◽  
Green Fluorescent

ABSTRACT The growth activity of Pseudomonas putida cells colonizing the rhizosphere of barley seedlings was estimated at the single-cell level by monitoring ribosomal contents and synthesis rates. Ribosomal synthesis was monitored by using a system comprising a fusion of the ribosomal Escherichia coli rrnBP1 promoter to a gene encoding an unstable variant of the green fluorescent protein (Gfp). Gfp expression in a P. putida strain carrying this system inserted into the chromosome was strongly dependent on the growth phase and growth rate of the strain, and cells growing exponentially at rates of ≥0.17 h−1 emitted growth rate-dependent green fluorescence detectable at the single-cell level. The single-cell ribosomal contents were very heterogeneous, as determined by quantitative hybridization with fluorescently labeled rRNA probes in P. putida cells extracted from the rhizosphere of 1-day-old barley seedlings grown under sterile conditions. After this, cells extracted from the root system had ribosomal contents similar to those found in starved cells. There was a significant decrease in the ribosomal content of P. putida cells when bacteria were introduced into nonsterile bulk or rhizosphere soil, and the Gfp monitoring system was not induced in cells extracted from either of the two soil systems. The monitoring system used permitted nondestructive in situ detection of fast-growing bacterial microcolonies on the sloughing root sheath cells of 1- and 2-day-old barley seedlings grown under sterile conditions, which demonstrated that it may be possible to use the unstable Gfp marker for studies of transient gene expression in plant-microbe systems.

scholarly journals An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 60 ◽  
Author(s):  
Shin-ichiro Kojima ◽  
Gary G. Borisy
Keyword(s):  
Single Cell ◽  
Fluorescent Protein ◽  
Selection Marker ◽  
Target Sequence ◽  
Fluorescence Intensity Ratio ◽  
Single Cell Level ◽  
Reporter Assay ◽  
Red Fluorescent Protein ◽  
Dual Fluorescence ◽  
Cell Level

RNA interference (RNAi) is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA) in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA) together with an enhanced green fluorescent protein (EGFP); the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry). Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls) is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3) and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB) method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

In Situ Detection of High Levels of Horizontal Plasmid Transfer in Marine Bacterial Communities

1998 ◽  
Vol 64 (7) ◽  
pp. 2670-2675 ◽  
Author(s):  
Cecilia Dahlberg ◽  
Maria Bergström ◽  
Malte Hermansson
Keyword(s):  
Gene Transfer ◽  
Pseudomonas Putida ◽  
Single Cell ◽  
Fluorescent Protein ◽  
Detection System ◽  
Plasmid Transfer ◽  
Conjugative Plasmid ◽  
Epifluorescence Microscopy ◽  
Single Cell Level ◽  
Cell Level

ABSTRACT Gene transfer of the conjugative plasmid pBF1 fromPseudomonas putida to indigenous bacteria in seawater was investigated with a detection system for gene transfer based on the green fluorescent protein (GFP) (C. Dahlberg et al., Mol. Biol. Evol. 15:385–390, 1998). pBF1 was tagged with the gfp gene controlled by a lac promoter which is down regulated in the donor cell by a chromosomal repressor (lacI q). The plasmid donor cells (Pseudomonas putida KT2442) subsequently do not express gfp. Transfer to recipient strains lacking the repressor results in expression of gfp. The transconjugant can subsequently be detected by epifluorescence microscopy on a single-cell level. By using this method, transfer of pBF1::gfp and expression of the gfpgene were first shown to occur during nutrient-limiting conditions to several defined recipient bacteria in artificial seawater. Second, we measured transfer of pBF1 from P. putida to the marine bacterial community directly in seawater samples, on a single-cell level, without limiting the detection of gene transfer to the culturable fraction of bacteria. Plasmid transfer was detected on surfaces and in bulk seawater. Seawater bacteria with different morphologies were shown to receive the plasmid. Gene transfer frequencies of 2.3 × 10−6 to 2.2 × 10−4 transconjugants per recipient were recorded after 3 days of incubation.

scholarly journals High Sensitivity In Vivo Imaging of Cancer Metastasis Using a Near-Infrared Luciferin Analogue seMpai

2020 ◽  
Vol 21 (21) ◽  
pp. 7896
Author(s):  
Jun Nakayama ◽  
Ryohei Saito ◽  
Yusuke Hayashi ◽  
Nobuo Kitada ◽  
Shota Tamaki ◽  
...  
Keyword(s):  
Single Cell ◽  
In Vivo Imaging ◽  
Cancer Metastasis ◽  
Near Infrared ◽  
High Sensitivity ◽  
Single Cell Level ◽  
Background Signal ◽  
Cell Level ◽  
Highly Sensitive

Bioluminescence imaging (BLI) is useful to monitor cell movement and gene expression in live animals. However, D-luciferin has a short wavelength (560 nm) which is absorbed by tissues and the use of near-infrared (NIR) luciferin analogues enable high sensitivity in vivo BLI. The AkaLumine-AkaLuc BLI system (Aka-BLI) can detect resolution at the single-cell level; however, it has a clear hepatic background signal. Here, to enable the highly sensitive detection of bioluminescence from the surrounding liver tissues, we focused on seMpai (C15H16N3O2S) which has been synthesized as a luciferin analogue and has high luminescent abilities as same as AkaLumine. We demonstrated that seMpai BLI could detect micro-signals near the liver without any background signal. The solution of seMpai was neutral; therefore, seMpai imaging did not cause any adverse effect in mice. seMpai enabled a highly sensitive in vivo BLI as compared to previous techniques. Our findings suggest that the development of a novel mutated luciferase against seMpai may enable a highly sensitive BLI at the single-cell level without any background signal. Novel seMpai BLI system can be used for in vivo imaging in the fields of life sciences and medicine.

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