scholarly journals Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system

2019 ◽  
Vol 116 (52) ◽  
pp. 26491-26496 ◽  
Author(s):  
Carola Gregor ◽  
Jasmin K. Pape ◽  
Klaus C. Gwosch ◽  
Tanja Gilat ◽  
Steffen J. Sahl ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Bioluminescence Imaging ◽  
Single Cells ◽  
Cell Types ◽  
Firefly Luciferase ◽  
Living Cells ◽  
Spatiotemporal Resolution ◽  
Bacterial Bioluminescence ◽  
Mammalian Cell Lines ◽  
Bioluminescence Signal

Bioluminescence-based imaging of living cells has become an important tool in biological and medical research. However, many bioluminescence imaging applications are limited by the requirement of an externally provided luciferin substrate and the low bioluminescence signal which restricts the sensitivity and spatiotemporal resolution. The bacterial bioluminescence system is fully genetically encodable and hence produces autonomous bioluminescence without an external luciferin, but its brightness in cell types other than bacteria has, so far, not been sufficient for imaging single cells. We coexpressed codon-optimized forms of the bacterialluxCDABEandfrpgenes from multiple plasmids in different mammalian cell lines. Our approach produces high luminescence levels that are comparable to firefly luciferase, thus enabling autonomous bioluminescence microscopy of mammalian cells.

scholarly journals Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system

2019 ◽  
Author(s):  
Carola Gregor ◽  
Jasmin K. Pape ◽  
Klaus C. Gwosch ◽  
Tanja Gilat ◽  
Steffen J. Sahl ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Bioluminescence Imaging ◽  
Single Cells ◽  
Cell Types ◽  
Firefly Luciferase ◽  
Host Cells ◽  
Spatiotemporal Resolution ◽  
Fungal Host ◽  
Bacterial Bioluminescence ◽  
Mammalian Cell Lines

AbstractBioluminescence based imaging of living cells has become an important tool in biological and medical research. However, many bioluminescence imaging applications are limited by the requirement of an externally provided luciferin substrate and the low bioluminescence signal which restricts the sensitivity and spatiotemporal resolution. The bacterial bioluminescence system is fully genetically encodable and hence produces autonomous bioluminescence without an external luciferin, but its brightness in cell types other than bacteria has so far not been sufficient for imaging single cells. We coexpressed codon-optimized forms of the bacterial luxCDABE and frp genes from multiple plasmids in different mammalian cell lines. Our approach produces high luminescence levels that are comparable to firefly luciferase, thus enabling autonomous bioluminescence microscopy of mammalian cells.Significance statementBioluminescence is generated by luciferases that oxidize a specific luciferin. The enzymes involved in the synthesis of the luciferin from widespread cellular metabolites have so far been identified for only two bioluminescence systems, those of bacteria and fungi. In these cases, the complete reaction cascade is genetically encodable, meaning that heterologous expression of the corresponding genes can potentially produce autonomous bioluminescence in cell types other than the bacterial or fungal host cells. However, the light levels achieved in mammalian cells so far are not sufficient for single-cell applications. Here we present, for the first time, autonomous bioluminescence images of single mammalian cells by coexpression of the genes encoding the six enzymes from the bacterial bioluminescence system.

scholarly journals In Vivo Bioluminescence Imaging for the Study of Intestinal Colonization by Escherichia coli in Mice

2009 ◽  
Vol 76 (1) ◽  
pp. 264-274 ◽  
Author(s):  
M.-L. Foucault ◽  
L. Thomas ◽  
S. Goussard ◽  
B. R. Branchini ◽  
C. Grillot-Courvalin
Keyword(s):  
Escherichia Coli ◽  
Bioluminescence Imaging ◽  
Light Emission ◽  
Direct Method ◽  
Firefly Luciferase ◽  
Intestinal Colonization ◽  
Bacterial Populations ◽  
Bioluminescence Signal

ABSTRACT Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R 2 = 0.98) or transcutaneously in the abdominal region of whole animals (R 2 = 0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2ΩPamiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.

scholarly journals Reticulon 4a promotes exocytosis in mammalian cells

2019 ◽  
Vol 30 (18) ◽  
pp. 2349-2357 ◽  
Author(s):  
Richik Nilay Mukherjee ◽  
Daniel L. Levy
Keyword(s):  
Cell Surface ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Cell Types ◽  
Vesicular Trafficking ◽  
Secreted Proteins ◽  
Mammalian Cell Lines ◽  
Rough Er ◽  
Different Cell Types ◽  
Functional Output

Endoplasmic reticulum (ER) tubules and sheets conventionally correspond to smooth and rough ER, respectively. The ratio of ER tubules-to-sheets varies in different cell types and changes in response to cellular conditions, potentially impacting the functional output of the ER. To directly test whether ER morphology impacts vesicular trafficking, we increased the tubule-to-sheet ratio in three different ways, by overexpressing Rtn4a, Rtn4b, or REEP5. Only Rtn4a overexpression increased exocytosis, but not overall levels, of several cell surface and secreted proteins. Furthermore, Rtn4a depletion reduced cell surface trafficking without affecting ER morphology. Similar results were observed in three different mammalian cell lines, suggesting that Rtn4a generally enhances exocytosis independently of changes in ER morphology. Finally, we show that Rtn4a levels modulate cell adhesion, possibly by regulating trafficking of integrins to the cell surface. Taking the results together, we find that altering ER morphology does not necessarily affect protein trafficking, but that Rtn4a specifically enhances exocytosis.

scholarly journals Polarization of fluorescein fluorescence in single cells.

1979 ◽  
Vol 27 (1) ◽  
pp. 49-55 ◽  
Author(s):  
R Udkoff ◽  
A Norman
Keyword(s):  
Mammalian Cells ◽  
Single Cells ◽  
Classical Model ◽  
Cell Types ◽  
Wavelength Shift ◽  
Two Peaks ◽  
Vitro Data ◽  
P Distribution ◽  
In Vitro Data

Measurement of fluorescence polarization (P) gives information about the immediate environment of the fluorescent molecule. We used a flow polarimeter to investigate the factors influencing P of fluorescein in mammalian cells to determine whether such measurements are useful for characterizing heterogeneous cell populations. Fluorescein was introduced into cells by incubation with FDA. Measurements of the intensity of fluorescence (TI) and polarization (P) revealed an unexpected dependence: P decreased with increasing intensity of fluorescence. This may be accounted for by the classical model of the binding of small molecules to protein in which P is dependent on the ratio bound to unbound molecules. We have been able to estimate the quenching due to binding and construct a Scatchard plot. We estimated a wavelength shift from in vitro data consistent with the dependence of P on wavelength seen in our cell work. Generally, the distributions of P are symmetrical. Photon statistics broadens the P distribution of dim cells. However, structure does develop in the P distribution when the cells are deprived of calcium or incubated in the cold. This appears as a shoulder on the P distribution or resolves into two peaks. Calcium deprivation may differentially affect a subpopulation of cells whose significance remains to be explored in various cell types.

scholarly journals Actin dynamics in living mammalian cells

1998 ◽  
Vol 111 (12) ◽  
pp. 1649-1658 ◽  
Author(s):  
C. Ballestrem ◽  
B. Wehrle-Haller ◽  
B.A. Imhof
Keyword(s):  
Actin Cytoskeleton ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Time Lapse ◽  
Living Cells ◽  
Actin Dynamics ◽  
Mammalian Cell Lines ◽  
Cytoskeletal Dynamics ◽  
Green Fluorescent

The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human β-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured ‘actin clouds’ were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.

scholarly journals Strongly enhanced bacterial bioluminescence with the ilux operon for single-cell imaging

2018 ◽  
Vol 115 (5) ◽  
pp. 962-967 ◽  
Author(s):  
Carola Gregor ◽  
Klaus C. Gwosch ◽  
Steffen J. Sahl ◽  
Stefan W. Hell
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Flavin Mononucleotide ◽  
Background Signal ◽  
Cell Level ◽  
Spatiotemporal Resolution ◽  
High Background ◽  
E Coli ◽  
Bacterial Bioluminescence ◽  
Single Cell Imaging

Bioluminescence imaging of single cells is often complicated by the requirement of exogenous luciferins that can be poorly cell-permeable or produce high background signal. Bacterial bioluminescence is unique in that it uses reduced flavin mononucleotide as a luciferin, which is abundant in all cells, making this system purely genetically encodable by the lux operon. Unfortunately, the use of bacterial bioluminescence has been limited by its low brightness compared with other luciferases. Here, we report the generation of an improved lux operon named ilux with an approximately sevenfold increased brightness when expressed in Escherichia coli; ilux can be used to image single E. coli cells with enhanced spatiotemporal resolution over several days. In addition, since only metabolically active cells produce bioluminescent signal, we show that ilux can be used to observe the effect of different antibiotics on cell viability on the single-cell level.

scholarly journals Simultaneous quantification of protein-DNA contacts and transcriptomes in single cells

2019 ◽  
Author(s):  
Koos Rooijers ◽  
Corina M. Markodimitraki ◽  
Franka J. Rang ◽  
Sandra S. de Vries ◽  
Alex Chialastri ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Single Cells ◽  
Critical Role ◽  
Nuclear Lamina ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Expression Variability ◽  
The Impact

AbstractThe epigenome plays a critical role in regulating gene expression in mammalian cells. However, understanding how cell-to-cell heterogeneity in the epigenome influences gene expression variability remains a major challenge. Here we report a novel method for simultaneous single-cell quantification of protein-DNA contacts with DamID and transcriptomics (scDamID&T). This method enables quantifying the impact of protein-DNA contacts on gene expression from the same cell. By profiling lamina-associated domains (LADs) in human cells, we reveal different dependencies between genome-nuclear lamina (NL) association and gene expression in single cells. In addition, we introduce the E. coli methyltransferase, Dam, as an in vivo marker of chromatin accessibility in single cells and show that scDamID&T can be utilized as a general technology to identify cell types in silico while simultaneously determining the underlying gene-regulatory landscape. With this strategy the effect of chromatin states, transcription factor binding, and genome organization on the acquisition of cell-type specific transcriptional programs can be quantified.

scholarly journals Mammalian cells stably overexpressing N-acylphosphatidylethanolamine-hydrolysing phospholipase D exhibit significantly decreased levels of N-acylphosphatidylethanolamines

2005 ◽  
Vol 389 (1) ◽  
pp. 241-247 ◽  
Author(s):  
Yasuo OKAMOTO ◽  
Jun MORISHITA ◽  
Jun WANG ◽  
Patricia C. SCHMID ◽  
Randy J. KREBSBACH ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Phospholipase D ◽  
Mammalian Cells ◽  
Fold Increase ◽  
Living Cells ◽  
Animal Tissues ◽  
Hek 293 ◽  
Transfected Cells ◽  
Mammalian Cell Lines

In animal tissues, NAEs (N-acylethanolamines), including N-arachidonoylethanolamine (anandamide), are primarily formed from their corresponding NAPEs (N-acylphosphatidylethanolamines) by a phosphodiesterase of the PLD (phospholipase D) type (NAPE-PLD). Recently, we cloned cDNAs of NAPE-PLD from mouse, rat and human [Okamoto, Morishita, Tsuboi, Tonai and Ueda (2004) J. Biol. Chem. 279, 5298–5305]. However, it remained unclear whether NAPE-PLD acts on endogenous NAPEs contained in the membrane of living cells. To address this question, we stably transfected two mammalian cell lines (HEK-293 and CHO-K1) with mouse NAPE-PLD cDNA, and investigated the endogenous levels and compositions of NAPEs and NAEs in these cells, compared with mock-transfected cells, with the aid of GC-MS. The overexpression of NAPE-PLD caused a decrease in the total amount of NAPEs by 50–90% with a 1.5-fold increase in the total amount of NAEs, suggesting that the recombinant NAPE-PLD utilizes endogenous NAPE as a substrate in the cell. Since the compositions of NAEs and NAPEs of NAPE-PLD-overexpressing cells and mock-transfected cells were very similar, the enzyme did not appear to discriminate among the N-acyl groups of endogenous NAPEs. These results confirm that overexpressed NAPE-PLD is capable of forming NAEs, including anandamide, in living cells.

scholarly journals Genetic screens in isogenic mammalian cell lines without single cell cloning

2019 ◽  
Author(s):  
Peter C DeWeirdt ◽  
Kendall R Sanson ◽  
Ruth E Hanna ◽  
Mudra Hegde ◽  
Annabel K Sangree ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Genetic Interaction ◽  
Cell Types ◽  
Repair Gene ◽  
Synthetic Lethal ◽  
Mammalian Cell Lines ◽  
Genome Wide ◽  
Single Cell Cloning ◽  
Synthetic Lethal Interactions

Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs for mammalian cells, however, is labor-intensive, time-consuming, and impossible in some cell types. Here we present an approach to create isogenic pairs of cells and screen them with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We queried the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, via 25 genome-wide screens across 4 cell lines. For all three genes, we identify a rich set of both expected and novel buffering and synthetic lethal interactions. Further, we compare the interactions observed in genetic space to those found when targeting these genes with small molecules and identify hits that may inform the clinical uses for these inhibitors. We anticipate that this methodology will be broadly useful to comprehensively study genes of interest across many cell types.

scholarly journals Single-cell nucleic acid profiling in droplets (SNAPD) enables high-throughput analysis of heterogeneous cell populations

2020 ◽  
Author(s):  
Leland B. Hyman ◽  
Clare R. Christopher ◽  
Philip A. Romero
Keyword(s):  
Nucleic Acid ◽  
Single Cell ◽  
Mammalian Cells ◽  
Cancer Biology ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
High Throughput Analysis

AbstractExperimental methods that capture the individual properties of single cells are revealing the key role of cell-to-cell variability in countless biological processes. These single-cell methods are becoming increasingly important across the life sciences in fields such as immunology, regenerative medicine, and cancer biology. Existing single-cell analysis methods are often limited by their low analysis throughput, their inability to profile high-dimensional phenotypes, and complicated experimental workflows with slow turnaround times. In this work, we present Single-cell Nucleic Acid Profiling in Droplets (SNAPD) to analyze the transcriptional states of hundreds of thousands of single mammalian cells. Individual cells are encapsulated in aqueous droplets on a microfluidic chip and the content of each cell is profiled by amplifying a targeted panel of transcriptional markers. Molecular logic circuits then integrate this multi-dimensional information to categorize cells based on their transcriptional profile and produce a detectable fluorescence output. SNAPD analyzes over 100,000 cells per hour and can be used to quantify distinct cell types within populations, detect rare cells at frequencies down to 0.1%, and enrich specific cell types using microfluidic sorting. SNAPD provides a simple, rapid, low cost, and scalable approach to study complex phenotypes in heterogeneous cell populations.

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