Single-molecule mRNA and translation imaging in neurons

2021 ◽  
Author(s):  
Jessica Mitchell ◽  
Jeffrey A. Chao
Keyword(s):  
Single Molecule ◽  
Neuronal Plasticity ◽  
Behavioural Change ◽  
Live Cells ◽  
Local Translation ◽  
Single Molecule Fluorescence ◽  
Technical Developments ◽  
Mrna Transcripts ◽  
Neuronal Compartments

Memory-relevant neuronal plasticity is believed to require local translation of new proteins at synapses. Understanding this process has necessitated the development of tools to visualize mRNA within relevant neuronal compartments. In this review, we summarize the technical developments that now enable mRNA transcripts and their translation to be visualized at single-molecule resolution in both fixed and live cells. These tools include single-molecule fluorescence in situ hybridization (smFISH) to visualize mRNA in fixed cells, MS2/PP7 labelling for live mRNA imaging and SunTag labelling to observe the emergence of nascent polypeptides from a single translating mRNA. The application of these tools in cultured neurons and more recently in whole brains promises to revolutionize our understanding of local translation in the neuronal plasticity that underlies behavioural change.

scholarly journals Selective dendritic localization of mRNA in Drosophila mushroom body output neurons

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jessica Mitchell ◽  
Carlas S Smith ◽  
Josh Titlow ◽  
Nils Otto ◽  
Pieter van Velde ◽  
...  
Keyword(s):  
Single Molecule ◽  
Nicotinic Acetylcholine Receptors ◽  
Neuronal Plasticity ◽  
Mushroom Body ◽  
Acetylcholine Receptors ◽  
Behavioural Change ◽  
Before And After ◽  
Specific Mrnas ◽  
Output Neurons ◽  
Subcellular Resolution

Memory-relevant neuronal plasticity is believed to require local translation of new proteins at synapses. Understanding this process requires the visualization of the relevant mRNAs within these neuronal compartments. Here we used single-molecule fluorescence in situ hybridization (smFISH) to localize mRNAs at subcellular resolution in the adult Drosophila brain. mRNAs for subunits of nicotinic acetylcholine receptors and kinases could be detected within the dendrites of co-labelled Mushroom Body Output Neurons (MBONs) and their relative abundance showed cell-specificity. Moreover, aversive olfactory learning produced a transient increase in the level of CaMKII mRNA within the dendritic compartments of the 52a MBONs. Localization of specific mRNAs in MBONs before and after learning represents a critical step towards deciphering the role of dendritic translation in the neuronal plasticity underlying behavioural change in Drosophila.

scholarly journals In situ genotyping of a pooled strain library after characterizing complex phenotypes

2017 ◽  
Author(s):  
Michael J. Lawson ◽  
Daniel Camsund ◽  
Jimmy Larsson ◽  
Özden Baltekin ◽  
David Fange ◽  
...  
Keyword(s):  
Fluorescence Imaging ◽  
Single Molecule ◽  
Single Molecule Fluorescence ◽  
Detailed Characterization ◽  
E Coli ◽  
Complex Phenotypes

So far, it has not been possible to perform advanced microscopy on pool generated strain libraries and at the same time know each strain’s genotype. We have overcome this barrier by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single molecule fluorescence imaging of E. coli strains harboring barcoded plasmids that express a sgRNA which suppress different genes through dCas9.

scholarly journals Alpha-satellite RNA transcripts are repressed by centromere–nucleolus associations

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Leah Bury ◽  
Brittania Moodie ◽  
Jimmy Ly ◽  
Liliana S McKay ◽  
Karen HH Miga ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Single Molecule ◽  
Alpha Satellite ◽  
Satellite Rna ◽  
Single Molecule Fluorescence ◽  
Chromatin Dynamics ◽  
Rna Transcripts ◽  
Non Coding Rnas

Although originally thought to be silent chromosomal regions, centromeres are instead actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA-smFISH foci levels vary across cell lines and over the cell cycle, but do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Alpha-satellite expression occurs through RNA polymerase II-dependent transcription, but does not require established centromere or cell division components. Instead, our work implicates centromere–nucleolar interactions as repressing alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels across cell lines and transcript levels increase substantially when the nucleolus is disrupted. The control of alpha-satellite transcripts by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions.

scholarly journals Identification of multiple kinetic populations of DNA-binding proteins in live cells

2019 ◽  
Author(s):  
Han N. Ho ◽  
Daniel Zalami ◽  
Jürgen Köhler ◽  
Antoine M. van Oijen ◽  
Harshad Ghodke
Keyword(s):  
Dna Binding ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
Binding Proteins ◽  
Dynamic Range ◽  
Time Lapse ◽  
Dna Binding Proteins ◽  
Live Cells ◽  
Single Molecule Fluorescence ◽  
Time Lapse Imaging

ABSTRACTUnderstanding how multi-protein complexes function in cells requires detailed quantitative understanding of their association and dissociation kinetics. Analysis of the heterogeneity of binding lifetimes enables interrogation of the various intermediate states formed during the reaction. Single-molecule fluorescence imaging permits the measurement of reaction kinetics inside living organisms with minimal perturbation. However, poor photo-physical properties of fluorescent probes limit the dynamic range and accuracy of measurements of off rates in live cells. Time-lapse single-molecule fluorescence imaging can partially overcome the limits of photobleaching, however, limitations of this technique remain uncharacterized. Here, we present a structured analysis of which timescales are most accessible using the time-lapse imaging approach and explore uncertainties in determining kinetic sub-populations. We demonstrate the effect of shot noise on the precision of the measurements, as well as the resolution and dynamic range limits that are inherent to the method. Our work provides a convenient implementation to determine theoretical errors from measurements and to support interpretation of experimental data.STATEMENT OF SIGNIFICANCEMeasuring lifetimes of interactions between DNA-binding proteins and their substrates is important for understanding how they function in cells. In principle, time-lapse imaging of fluorescently-tagged proteins using single-molecule methods can be used to identify multiple sub-populations of DNA-binding proteins and determine binding lifetimes lasting for several tens of minutes. Despite this potential, currently available guidelines for the selection of binding models are unreliable, and the practical implementation of this approach is limited. Here, using experimental and simulated data we identify the minimum size of the dataset required to resolve multiple populations reliably and measure binding lifetimes with desired accuracy. This work serves to provide a guide to data collection, and measurement of DNA-binding lifetimes from single-molecule time-lapse imaging data.

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