scholarly journals Early alphavirus replication dynamics in single cells reveal a passive basis for superinfection exclusion

2020 ◽  
Author(s):  
Zakary S. Singer ◽  
Pradeep M. Ambrose ◽  
Tal Danino ◽  
Charles M. Rice
Keyword(s):  
Single Molecule ◽  
Time Window ◽  
Single Cells ◽  
Viral Kinetics ◽  
Protein Kinetics ◽  
Superinfection Exclusion ◽  
Viral Replicase ◽  
Competitive Phenotypes ◽  
Over Time

SummaryWhile decades of research have elucidated many steps in the alphavirus lifecycle, the earliest replication dynamics have remained unclear. This missing time window has obscured early replicase strand synthesis behavior and prevented elucidation of how the resulting activity gives rise to a superinfection exclusion environment, one of the fastest competitive phenotypes among viruses. Using quantitative live-cell and single-molecule imaging, we characterize the strand preferences of the viral replicase in situ, and measure protein kinetics in single cells over time. In this framework, we evaluate competition between alphaviruses, and uncover that early superinfection exclusion is actually not a binary and unidirectional process, but rather a graded and bidirectional viral interaction. In contrast to competition between other viruses, alphaviruses demonstrate a passive basis for superinfection exclusion, emphasizing the utility of analyzing viral kinetics within single cells.

scholarly journals mRNA detection in budding yeast with single fluorophores

2017 ◽  
Author(s):  
Gable M. Wadsworth ◽  
Rasesh Y. Parikh ◽  
John S. Choy ◽  
Harold D. Kim
Keyword(s):  
Single Molecule ◽  
Budding Yeast ◽  
Single Cells ◽  
Phenotypic Variability ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Mrna Levels ◽  
Protein Levels ◽  
Mrna Isoform

Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells. Single-molecule mRNA Fluorescence In Situ Hybridization (FISH) has been established as the standard method for this purpose, but current protocols require a long region of mRNA to be targeted by multiple DNA probes. Here, we introduce a new single-probe FISH protocol termed sFISH for budding yeast, Saccharomyces cerevisiae using a single DNA probe labeled with a single fluorophore. In sFISH, we markedly improved probe specificity and signal-to-background ratio by using methanol fixation and inclined laser illumination. We show that sFISH reports mRNA changes that correspond to protein levels and gene copy number. Using this new FISH protocol, we can detect more than 50% of the total target mRNA. We also demonstrate the versatility of sFISH using FRET detection and mRNA isoform profiling as examples. Our FISH protocol with single-fluorophore sensitivity significantly reduces cost and time compared to the conventional FISH protocols and opens up new opportunities to investigate small changes in RNA at the single cell level.

scholarly journals Multiplex labeling and manipulation of endogenous neuronal proteins using sequential CRISPR/Cas9 gene editing

2022 ◽  
Author(s):  
Wouter J Droogers ◽  
Jelmer Willems ◽  
Harold D MacGillavry ◽  
Arthur PH de Jong
Keyword(s):  
Genome Editing ◽  
Single Molecule ◽  
Time Window ◽  
Single Cells ◽  
Synaptic Proteins ◽  
Flp Recombinase ◽  
Neuronal Proteins ◽  
Endogenous Proteins ◽  
Living Neurons ◽  
High Degree

Recent advances in CRISPR/Cas9-mediated knock-in methods enable labeling of individual endogenous proteins with fluorophores, to determine their spatiotemporal expression in intact biological preparations. However, multiplex knock-in methods remain limited, particularly in postmitotic cells, due to a high degree of crosstalk between genome editing events. We present Conditional Activation of Knock-in Expression (CAKE), which delivers efficient, flexible and accurate multiplex genome editing in neurons. CAKE is based on sequential gRNA expression operated by a Cre- or Flp-recombinase to control the time window for genomic integration of each donor sequence, which diminishes crosstalk between genome editing events. Importantly, CAKE is compatible with multiple CRISPR/Cas9 strategies, and we show the utilization of CAKE for co-localization of various endogenous proteins, including synaptic scaffolds, ion channels and neurotransmitter receptor subunits. Knock-in efficacy was highly sensitive to DNA vector amount, while knock-in crosstalk was dependent on the rate of donor DNA integration and timing of Cre activation. We applied CAKE to study the co-distribution of endogenous synaptic proteins using dual-color single-molecule localization microscopy, and we introduced dimerization modules to acutely control synaptic receptor dynamics in living neurons. Taken together, CAKE is a versatile method for multiplex protein labeling, enabling accurate detection, precise localization and acute manipulation of endogenous proteins in single cells.

scholarly journals smiFISH and embryo segmentation for single-cell multi-gene RNA quantification in arthropods

2020 ◽  
Author(s):  
Llilians Calvo ◽  
Matthew Ronshaugen ◽  
Tom Pettini
Keyword(s):  
In Situ Hybridization ◽  
Single Cell ◽  
Single Molecule ◽  
Fluorescent In Situ Hybridization ◽  
Single Cells ◽  
Fano Factor ◽  
Confocal Imaging ◽  
Cell Segmentation ◽  
Expression Variability

ABSTRACTRecently, advances in fluorescent in-situ hybridization techniques and in imaging technology have enabled visualisation and counting of individual RNA molecules in single cells. This has greatly enhanced the resolution in our understanding of transcriptional processes. Here, we adapt a recently published smiFISH protocol (single-molecule inexpensive fluorescent in-situ hybridization) to whole embryos across a range of arthropod model species, and also to non-embryonic tissues. Using multiple fluorophores with distinct spectra and white light laser confocal imaging, we simultaneously detect and separate single RNAs from up to eight different genes in a whole embryo. We also combine smiFISH with cell membrane immunofluorescence, and present an imaging and analysis pipeline for 3D cell segmentation and single-cell RNA counting in whole blastoderm embryos. Finally, using whole embryo single-cell RNA count data, we propose two alternative single-cell variability measures to the commonly used Fano factor, and compare the capacity of these three measures to address different aspects of single-cell expression variability.

scholarly journals smiFISH and embryo segmentation for single-cell multi-gene RNA quantification in arthropods

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Llilians Calvo ◽  
Matthew Ronshaugen ◽  
Tom Pettini
Keyword(s):  
In Situ Hybridization ◽  
Single Cell ◽  
Single Molecule ◽  
Fluorescent In Situ Hybridization ◽  
Single Cells ◽  
Fano Factor ◽  
Confocal Imaging ◽  
Cell Segmentation ◽  
Expression Variability

AbstractRecently, advances in fluorescent in-situ hybridization techniques and in imaging technology have enabled visualization and counting of individual RNA molecules in single cells. This has greatly enhanced the resolution in our understanding of transcriptional processes. Here, we adapt a recently published smiFISH protocol (single-molecule inexpensive fluorescent in-situ hybridization) to whole embryos across a range of arthropod model species, and also to non-embryonic tissues. Using multiple fluorophores with distinct spectra and white light laser confocal imaging, we simultaneously detect and separate single RNAs from up to eight different genes in a whole embryo. We also combine smiFISH with cell membrane immunofluorescence, and present an imaging and analysis pipeline for 3D cell segmentation and single-cell RNA counting in whole blastoderm embryos. Finally, using whole embryo single-cell RNA count data, we propose two alternative single-cell variability measures to the commonly used Fano factor, and compare the capacity of these three measures to address different aspects of single-cell expression variability.

scholarly journals Enzyme-based synthesis of single molecule RNA FISH probes

2018 ◽  
Author(s):  
Christian Lanctôt
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Low Cost ◽  
Single Cells ◽  
Rna Sequences ◽  
Rna Fish ◽  
Terminal Deoxynucleotide Transferase ◽  
Target Rna ◽  
Subcellular Level

ABSTRACTSingle molecule RNA fluorescence in situ hybridization (smRNA FISH) allows the quantitative analysis of gene expression in single cells. The technique relies on the use of pools of end-labeled fluorescent oligonucleotides to detect specific cellular RNA sequences. These fluorescent probes are currently chemically synthesized. Here I describe a novel technique based on the use of routine molecular biology enzymes to generate smRNA FISH probes without the need for chemical synthesis of pools of oligonucleotides. The protocol comprises 3 main steps: purification of phagemid-derived single stranded DNA molecules comprising a segment complementary to a target RNA sequence; fragmentation of these molecules by limited DNase I digestion; and end-labeling of the resulting oligonucleotides with terminal deoxynucleotide transferase and fluorescent dideoxynucleotides. smRNA FISH probes that are obtained using the technique presented here are shown to perform as well as conventional probes. The main advantages of the method are the low cost of probes and the flexibility it affords in the choice of labels. Enzyme-based synthesis of probes should further increase the popularity of smRNA FISH as a tool to investigate gene expression at the cellular or subcellular level.

scholarly journals Temporal Changes in Microscale Colonization of the Phylloplane by Aureobasidium pullulans

2006 ◽  
Vol 72 (9) ◽  
pp. 6234-6241 ◽  
Author(s):  
Molly J. McGrath ◽  
John H. Andrews
Keyword(s):  
Aureobasidium Pullulans ◽  
Single Cells ◽  
Growing Season ◽  
Epifluorescence Microscopy ◽  
Hybridization Probe ◽  
Cell Numbers ◽  
Growing Seasons ◽  
Apple Leaves ◽  
Over Time

ABSTRACT Colonization of apple leaves by the yeastlike fungus Aureobasidium pullulans was followed quantitatively and spatially at a microscale level throughout two growing seasons. Ten field leaves were sampled on 11 dates in 2003 and 15 dates in 2004. Using an A. pullulans-specific fluorescence in situ hybridization probe and epifluorescence microscopy, we enumerated total cells, swollen-cells and chlamydospores (SCC), and blastospores/mm2 on leaf features, including the midvein, other (smaller) veins, and the interveinal regions. By 7 July 2003 and 7 June 2004, the total numbers of A. pullulans cells/mm2 were significantly higher (P < 0.05) on the midvein and other veins than in the interveinal regions. This pattern remained consistent thereafter. The primary colonizing morphotype in all regions at all dates was the SCC form, although blastospores always occurred in low numbers. Occupancy was quantified based on the percentage of microscope fields of a particular leaf feature containing ≥1 A. pullulans cell. In general, as seasons progressed, the percent occupancy of features increased and, for most midvein and veinal features, approximated 100% at the end of both growing seasons. Except for early collections, when A. pullulans cell numbers were low, the percent occupancy of interveinal regions was lower than that of the midvein or other veinal regions. A. pullulans was distributed primarily as single cells throughout the seasons in interveinal regions. On the midvein and other veins, colonies of ≥4 cells developed over time, and more cells occurred in colonies than as singletons by August. Our results demonstrate that A. pullulans primarily colonizes veins, where populations appear to increase by growth in situ. This pattern is established early in the growing season and persists.

scholarly journals miR-9a regulates levels of both rhomboid mRNA and protein in the early Drosophila melanogaster embryo

2021 ◽  
Author(s):  
Lorenzo Gallicchio ◽  
Sam Griffiths-Jones ◽  
Matthew Ronshaugen
Keyword(s):  
Drosophila Melanogaster ◽  
Single Cell ◽  
Single Molecule ◽  
Single Cells ◽  
Temporal Regulation ◽  
Cell Quantification ◽  
Early Embryos ◽  
Proliferation And Differentiation ◽  
Noise In Gene Expression

MicroRNAs have subtle and combinatorial effects on the expression levels of their targets. Studying the consequences of a single microRNA knockout often proves difficult as many such knockouts exhibit phenotypes only under stress conditions. This has led to the hypothesis that microRNAs frequently act as buffers of noise in gene expression. Observing and understanding buffering effects requires quantitative analysis of microRNA and target expression in single cells. To this end, we have employed single molecule fluorescence in situ hybridization, immunofluorescence, and high-resolution confocal microscopy to investigate the effects of miR-9a loss on the expression of the serine-protease rhomboid in Drosophila melanogaster early embryos. Our single-cell quantitative approach shows that rhomboid mRNA exhibits the same spatial expression pattern in WT and miR-9a knockout embryos, although the number of mRNA molecules per cell is higher when miR-9a is absent. However, the level of rhomboid protein shows a much more dramatic increase in the miR-9a> knockout. Specifically, we see accumulation of rhomboid protein in miR-9a mutants by stage 5, much earlier than in WT. The data therefore show that miR-9a functions in the regulation of rhomboid activity by both inducing mRNA degradation and inhibiting translation in the blastoderm embryo. Temporal regulation of neural proliferation and differentiation in vertebrates by miR-9a is well-established. We suggest that miR-9a family microRNAs are conserved regulators of timing in neurogenic processes. This work shows the power of single-cell quantification as an experimental tool to study phenotypic consequences of microRNA mis-regulation.

Optics-Free Visualization of Proteins in Single Cells by Time of Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags

2020 ◽  
Author(s):  
Feifei Jia ◽  
Jie Wang ◽  
Yanyan Zhang ◽  
Qun Luo ◽  
Luyu Qi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Secondary Ion Mass Spectrometry ◽  
Single Cells ◽  
Time Of Flight ◽  
E Coli ◽  
Tof Sims ◽  
Ion Mass Spectrometry ◽  
Chemical Tags ◽  
Secondary Ion

<p></p><p><i>In situ</i> visualization of proteins of interest at single cell level is attractive in cell biology, molecular biology and biomedicine, which usually involves photon, electron or X-ray based imaging methods. Herein, we report an optics-free strategy that images a specific protein in single cells by time of flight-secondary ion mass spectrometry (ToF-SIMS) following genetic incorporation of fluorine-containing unnatural amino acids as a chemical tag into the protein via genetic code expansion technique. The method was developed and validated by imaging GFP in E. coli and human HeLa cancer cells, and then utilized to visualize the distribution of chemotaxis protein CheA in E. coli cells and the interaction between high mobility group box 1 protein and cisplatin damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for <i>in situ </i>visualization of proteins of interest as well as the interactions between proteins and drugs or drug damaged DNA in single cells.</p><p></p>

In-Situ Dual Beam (FIBSEM) Techniques for Probe Pad Deposition and Dielectric Integrity Inspection in 0.2 μm Technology DRAM Single Cells

1999 ◽  
Author(s):  
Gunnar Zimmermann ◽  
Richard Chapman
Keyword(s):  
Electron Beam ◽  
Single Cell ◽  
Single Cells ◽  
Ion Beam ◽  
Gate Oxide ◽  
Ion Milling ◽  
Dual Beam ◽  
Sem Imaging ◽  
Innovative Techniques

Abstract Dual beam FIBSEM systems invite the use of innovative techniques to localize IC fails both electrically and physically. For electrical localization, we present a quick and reliable in-situ FIBSEM technique to deposit probe pads with very low parasitic leakage (Ipara &lt; 4E-11A at 3V). The probe pads were Pt, deposited with ion beam assistance, on top of highly insulating SiOx, deposited with electron beam assistance. The buried plate (n-Band), p-well, wordline and bitline of a failing and a good 0.2 μm technology DRAM single cell were contacted. Both cells shared the same wordline for direct comparison of cell characteristics. Through this technique we electrically isolated the fail to a single cell by detecting leakage between the polysilicon wordline gate and the cell diffusion. For physical localization, we present a completely in-situ FIBSEM technique that combines ion milling, XeF2 staining and SEM imaging. With this technique, the electrically isolated fail was found to be a hole in the gate oxide at the bad cell.

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