scholarly journals Quantitative analysis of chromatin compaction in living cells using FLIM–FRET

2009 ◽  
Vol 187 (4) ◽  
pp. 481-496 ◽  
Author(s):  
David Llères ◽  
John James ◽  
Sam Swift ◽  
David G. Norman ◽  
Angus I. Lamond
Keyword(s):  
Fluorescent Protein ◽  
Trichostatin A ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Human Cell Line ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Chromatin Compaction ◽  
Fret Efficiency ◽  
Green Fluorescent

We present a quantitative Förster resonance energy transfer (FRET)–based assay using multiphoton fluorescence lifetime imaging microscopy (FLIM) to measure chromatin compaction at the scale of nucleosomal arrays in live cells. The assay uses a human cell line coexpressing histone H2B tagged to either enhanced green fluorescent protein (FP) or mCherry FPs (HeLaH2B-2FP). FRET occurs between FP-tagged histones on separate nucleosomes and is increased when chromatin compacts. Interphase cells consistently show three populations of chromatin with low, medium, or high FRET efficiency, reflecting spatially distinct regions with different levels of chromatin compaction. Treatment with inhibitors that either increase chromatin compaction (i.e., depletion of adenosine triphosphate) or decrease chromosome compaction (trichostatin A) results in a parallel increase or decrease in the FLIM–FRET signal. In mitosis, the assay showed variation in compaction level, as reflected by different FRET efficiency populations, throughout the length of all chromosomes, increasing to a maximum in late anaphase. These data are consistent with extensive higher order folding of chromatin fibers taking place during anaphase.

scholarly journals HIV-1 Vpr Oligomerization but Not That of Gag Directs the Interaction between Vpr and Gag

2009 ◽  
Vol 84 (3) ◽  
pp. 1585-1596 ◽  
Author(s):  
Joëlle V. Fritz ◽  
Denis Dujardin ◽  
Julien Godet ◽  
Pascal Didier ◽  
Jan De Mey ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Physiology ◽  
Fluorescence Lifetime Imaging ◽  
M Cell ◽  
C Terminus ◽  
Green Fluorescent ◽  
Hiv 1

ABSTRACT During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral particles via an interaction with the C-terminal domain of the Gag polyprotein precursor Pr55Gag. Vpr has been implicated in the nuclear import of newly made viral DNA and subsequently in its transcription. In addition, Vpr can affect the cell physiology by causing G2/M cell cycle arrest and apoptosis. Vpr can form oligomers, but their roles have not yet been investigated. We have developed fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays to monitor the interaction between Pr55Gag and Vpr in HeLa cells. To that end, we used enhanced green fluorescent protein-Vpr that can be incorporated into the virus and tetracysteine (TC)-tagged Pr55Gag-TC. This TC motif is tethered to the C terminus of Pr55Gag and does not interfere with Pr55Gag trafficking and the assembly of virus-like particles (VLPs). Results show that the Pr55Gag-Vpr complexes accumulated mainly at the plasma membrane. In addition, results with Pr55Gag-TC mutants confirm that the 41LXXLF domain of Gag-p6 is essential for Pr55Gag-Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55Gag recognition and its accumulation at the plasma membrane. On the other hand, Pr55Gag-Vpr complexes are still formed when Pr55Gag carries mutations impairing its multimerization. These findings suggest that Pr55Gag-Vpr recognition and complex formation occur early during Pr55Gag assembly.

scholarly journals Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Yoon Namkung ◽  
Christian Le Gouill ◽  
Viktoria Lukashova ◽  
Hiroyuki Kobayashi ◽  
Mireille Hogue ◽  
...  
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
Protein Translocation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Specific Cell ◽  
Cell Compartments ◽  
Green Fluorescent ◽  
G Protein Coupled

Abstract Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes.

scholarly journals Genetically encoded ratiometric indicators for potassium ion

2018 ◽  
Author(s):  
Yi Shen ◽  
Sheng-Yi Wu ◽  
Vladimir Rancic ◽  
Yong Qian ◽  
Shin-Ichiro Miyashita ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Temporal Dynamics ◽  
Biological Activities ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Potassium Ion ◽  
Live Cells ◽  
Fret Efficiency

AbstractPotassium ion (K+) homeostasis and dynamics play critical roles in regulating various biological activities, and the ability to monitor K+ spatial-temporal dynamics is critical to understanding these biological functions. Here we report the design and characterization of a Förster resonance energy transfer (FRET)-based genetically encoded K+ indicator, KIRIN1, constructed by inserting a bacterial cytosolic K+ binding protein (Kbp) between a fluorescent protein (FP) FRET pair, mCerulean3 and cp173Venus. Binding of K+ induces a conformational change in Kbp, resulting in an increase in FRET efficiency. KIRIN1 was able to detect K+ at physiologically relevant concentrations in vitro and is highly selective toward K+ over Na+. We further demonstrated that KIRIN1 allowed real-time imaging of pharmacologically induced depletion of cytosolic K+ in live cells, and KIRIN1 also enabled optical tracing of K+ efflux and reuptake in neurons upon glutamate stimulation in cultured primary neurons. These results demonstrate that KIRIN1 is a valuable tool to detect K+in vitro and in live cells.

scholarly journals Fluorescent Protein-Based Autophagy Biosensors

Materials ◽  
2021 ◽  
Vol 14 (11) ◽  
pp. 3019
Author(s):  
Heejung Kim ◽  
Jihye Seong
Keyword(s):  
Energy Transfer ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Treatment Strategies ◽  
Resonance Energy ◽  
Live Cells ◽  
Spectral Profile ◽  
Spatiotemporal Resolution ◽  
Future Directions ◽  
Complex Process

Autophagy is an essential cellular process of self-degradation for dysfunctional or unnecessary cytosolic constituents and organelles. Dysregulation of autophagy is thus involved in various diseases such as neurodegenerative diseases. To investigate the complex process of autophagy, various biochemical, chemical assays, and imaging methods have been developed. Here we introduce various methods to study autophagy, in particular focusing on the review of designs, principles, and limitations of the fluorescent protein (FP)-based autophagy biosensors. Different physicochemical properties of FPs, such as pH-sensitivity, stability, brightness, spectral profile, and fluorescence resonance energy transfer (FRET), are considered to design autophagy biosensors. These FP-based biosensors allow for sensitive detection and real-time monitoring of autophagy progression in live cells with high spatiotemporal resolution. We also discuss future directions utilizing an optobiochemical strategy to investigate the in-depth mechanisms of autophagy. These cutting-edge technologies will further help us to develop the treatment strategies of autophagy-related diseases.

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 Kinesin-1 structural organization and conformational changes revealed by FRET stoichiometry in live cells

2007 ◽  
Vol 176 (1) ◽  
pp. 51-63 ◽  
Author(s):  
Dawen Cai ◽  
Adam D. Hoppe ◽  
Joel A. Swanson ◽  
Kristen J. Verhey
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Structural Organization ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Protein Activity ◽  
Kinesin Light Chain ◽  
Initial Interaction

Kinesin motor proteins drive the transport of cellular cargoes along microtubule tracks. How motor protein activity is controlled in cells is unresolved, but it is likely coupled to changes in protein conformation and cargo association. By applying the quantitative method fluorescence resonance energy transfer (FRET) stoichiometry to fluorescent protein (FP)–labeled kinesin heavy chain (KHC) and kinesin light chain (KLC) subunits in live cells, we studied the overall structural organization and conformation of Kinesin-1 in the active and inactive states. Inactive Kinesin-1 molecules are folded and autoinhibited such that the KHC tail blocks the initial interaction of the KHC motor with the microtubule. In addition, in the inactive state, the KHC motor domains are pushed apart by the KLC subunit. Thus, FRET stoichiometry reveals conformational changes of a protein complex in live cells. For Kinesin-1, activation requires a global conformational change that separates the KHC motor and tail domains and a local conformational change that moves the KHC motor domains closer together.

Sign in / Sign up

Export Citation Format

Share Document