scholarly journals Phasor Histone FLIM-FRET Microscopy Maps Nuclear-Wide Nanoscale Chromatin Architecture With Respect to Genetically Induced DNA Double-Strand Breaks

2021 ◽  
Vol 12 ◽  
Author(s):  
Jieqiong Lou ◽  
Ashleigh Solano ◽  
Zhen Liang ◽  
Elizabeth Hinde
Keyword(s):  
Chromatin Structure ◽  
Network Architecture ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Strand Break ◽  
Cell System ◽  
Fluorescence Lifetime Imaging ◽  
Dna Double Strand Breaks ◽  
Structural Framework ◽  
Intact Nucleus

A DNA double-strand break (DSB) takes place in the context of chromatin, and there is increasing evidence for chromatin structure to play a functional role in DSB signaling and repair. Thus, there is an emerging need for quantitative microscopy methods that can directly measure chromatin network architecture and detect changes in this structural framework upon DSB induction within an intact nucleus. To address this demand, here we present the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labeled histones in the DSB inducible via AsiSI cell system (DIvA), which has sufficient spatial resolution to map nuclear-wide chromatin compaction at the level of nucleosome proximity with respect to multiple site-specific DSBs. We also demonstrate that when phasor histone FLIM-FRET is coupled with immunofluorescence, this technology has the unique advantage of enabling exploration of any heterogeneity that exists in chromatin structure at the spatially distinct and genetically induced DSBs.

scholarly journals Ca2+-Dependent Protein Kinase 6 Enhances KAT2 Shaker Channel Activity in Arabidopsis thaliana

2021 ◽  
Vol 22 (4) ◽  
pp. 1596
Author(s):  
Elsa Ronzier ◽  
Claire Corratgé-Faillie ◽  
Frédéric Sanchez ◽  
Christian Brière ◽  
Tou Cheu Xiong
Keyword(s):  
Arabidopsis Thaliana ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Physical Interaction ◽  
Fluorescence Lifetime Imaging ◽  
Dependent Manner ◽  
In Planta ◽  
Knock Out ◽  
Calcium Dependent ◽  
Dependent Protein

Post-translational regulations of Shaker-like voltage-gated K+ channels were reported to be essential for rapid responses to environmental stresses in plants. In particular, it has been shown that calcium-dependent protein kinases (CPKs) regulate Shaker channels in plants. Here, the focus was on KAT2, a Shaker channel cloned in the model plant Arabidopsis thaliana, where is it expressed namely in the vascular tissues of leaves. After co-expression of KAT2 with AtCPK6 in Xenopuslaevis oocytes, voltage-clamp recordings demonstrated that AtCPK6 stimulates the activity of KAT2 in a calcium-dependent manner. A physical interaction between these two proteins has also been shown by Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM). Peptide array assays support that AtCPK6 phosphorylates KAT2 at several positions, also in a calcium-dependent manner. Finally, K+ fluorescence imaging in planta suggests that K+ distribution is impaired in kat2 knock-out mutant leaves. We propose that the AtCPK6/KAT2 couple plays a role in the homeostasis of K+ distribution in leaves.

scholarly journals Discs large 1 controls daughter-cell polarity after cytokinesis in vertebrate morphogenesis

2018 ◽  
Vol 115 (46) ◽  
pp. E10859-E10868 ◽  
Author(s):  
Yuwei Li ◽  
Jason A. Junge ◽  
Cosimo Arnesano ◽  
Garrett G. Gross ◽  
Jeffrey H. Miner ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Protein Interactions ◽  
Protein Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Explant Culture ◽  
Scaffolding Protein ◽  
Fluorescence Lifetime Imaging ◽  
Discs Large

Vertebrate embryogenesis and organogenesis are driven by cell biological processes, ranging from mitosis and migration to changes in cell size and polarity, but their control and causal relationships are not fully defined. Here, we use the developing limb skeleton to better define the relationships between mitosis and cell polarity. We combine protein-tagging and -perturbation reagents with advanced in vivo imaging to assess the role of Discs large 1 (Dlg1), a membrane-associated scaffolding protein, in mediating the spatiotemporal relationship between cytokinesis and cell polarity. Our results reveal that Dlg1 is enriched at the midbody during cytokinesis and that its multimerization is essential for the normal polarity of daughter cells. Defects in this process alter tissue dimensions without impacting other cellular processes. Our results extend the conventional view that division orientation is established at metaphase and anaphase and suggest that multiple mechanisms act at distinct phases of the cell cycle to transmit cell polarity. The approach employed can be used in other systems, as it offers a robust means to follow and to eliminate protein function and extends the Phasor approach for studying in vivo protein interactions by frequency-domain fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET) to organotypic explant culture.

Automated multiwell fluorescence lifetime imaging for Förster resonance energy transfer assays and high content analysis

2015 ◽  
Vol 7 (10) ◽  
pp. 4071-4089 ◽  
Author(s):  
Douglas J. Kelly ◽  
Sean C. Warren ◽  
Dominic Alibhai ◽  
Sunil Kumar ◽  
Yuriy Alexandrov ◽  
...  
Keyword(s):  
Content Analysis ◽  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
High Content Analysis ◽  
Förster Resonance

An HCA-FLIM instrument is presented alongside exemplar oligomerisation, intermolecular and intramolecular FRET assays that require robust measurement of small lifetime changes.

scholarly journals The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Erika Günther ◽  
André Klauß ◽  
Mauricio Toro-Nahuelpan ◽  
Dirk Schüler ◽  
Carsten Hille ◽  
...  
Keyword(s):  
Magnetic Particles ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Stimulated Emission ◽  
Fluorescence Lifetime Imaging ◽  
Sted Microscopy

AbstractProtein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy Förster resonance energy transfer (FLIM-FRET) and stimulated emission depletion (STED) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.

scholarly journals Spatial mapping of splicing factor complexes involved in exon and intron definition

2008 ◽  
Vol 181 (6) ◽  
pp. 921-934 ◽  
Author(s):  
Jonathan D. Ellis ◽  
David Llères ◽  
Marco Denegri ◽  
Angus I. Lamond ◽  
Javier F. Cáceres
Keyword(s):  
Rna Polymerase Ii ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Sr Proteins ◽  
Small Subunit ◽  
Spatial Mapping ◽  
Fluorescence Lifetime Imaging ◽  
Cell Nuclei ◽  
Exon Definition ◽  
Small Nuclear Ribonucleoprotein

We have analyzed the interaction between serine/arginine-rich (SR) proteins and splicing components that recognize either the 5′ or 3′ splice site. Previously, these interactions have been extensively characterized biochemically and are critical for both intron and exon definition. We use fluorescence resonance energy transfer (FRET) microscopy to identify interactions of individual SR proteins with the U1 small nuclear ribonucleoprotein (snRNP)–associated 70-kD protein (U1 70K) and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) in live-cell nuclei. We find that these interactions occur in the presence of RNA polymerase II inhibitors, demonstrating that they are not exclusively cotranscriptional. Using FRET imaging by means of fluorescence lifetime imaging microscopy (FLIM), we map these interactions to specific sites in the nucleus. The FLIM data also reveal a previously unknown interaction between HCC1, a factor related to U2AF65, with both subunits of U2AF. Spatial mapping using FLIM-FRET reveals differences in splicing factors interactions within complexes located in separate subnuclear domains.

scholarly journals Looking at the Functional State of Proteins Inside Cells

1999 ◽  
Vol 7 (8) ◽  
pp. 3-4
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Functional State ◽  
Spatial Data ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Cellular Functions ◽  
Lifetime Imaging ◽  
Intracellular Proteins

Many intracellular proteins are catalysts that regulate cellular functions. These catalysts can be assayed to determine their functional state, but untii now it was not possible to simultaneously obtain a functional analysis and spatial data. Tony Ng, Anthony Squire, and others, working in the laboratories of Phillippe Bastiaens and Peter Parker, have combined Fluorescence Lifetime Imaging Microscopy (FLIM) with Fluorescence Resonance Energy Transfer (FRET) to spatially resolve the activation of a protein catalyst within living cells. Their technique was also applied to fixed cells.

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