scholarly journals Spatiotemporal imaging of small GTPases activity in live cells

2016 ◽  
Vol 113 (50) ◽  
pp. 14348-14353 ◽  
Author(s):  
Stephanie Voss ◽  
Dennis M. Krüger ◽  
Oliver Koch ◽  
Yao-Wen Wu
Keyword(s):  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Specific Effector ◽  
Spatiotemporal Visualization ◽  
Cell Current

Ras-like small GTPases function as molecular switches and regulate diverse cellular events. To examine the dynamics of signaling requires spatiotemporal visualization of their activity in the cell. Current small GTPase sensors rely on specific effector domains that are available for only a small number of GTPases and compete for endogenous regulator/effector binding. Here, we describe versatile conformational sensors for GTPase activity (COSGAs) based on the conserved GTPase fold. Conformational changes upon GDP/GTP exchange were directly observed in solution, on beads, and in live cells by Förster resonance energy transfer (FRET). The COSGAs allow for monitoring of Rab1 and K-Ras activity in live cells using fluorescence lifetime imaging microscopy. We found that Rab1 is largely active in the cytoplasm and inactive at the Golgi, suggesting that the Golgi serves as the terminal of the Rab1 functional cycle. K-Ras displays polarized activity at the plasma membrane, with less activity at the edge of the cell and membrane ruffles.

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.

scholarly journals Bim escapes displacement by BH3-mimetic anti-cancer drugs by double-bolt locking both Bcl-XL and Bcl-2

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Qian Liu ◽  
Elizabeth J Osterlund ◽  
Xiaoke Chi ◽  
Justin Pogmore ◽  
Brian Leber ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Anti Cancer ◽  
Apoptotic Proteins ◽  
Binding Interfaces ◽  
Quantifying In ◽  
Bh3 Mimetic

Tumor initiation, progression and resistance to chemotherapy rely on cancer cells bypassing programmed cell death by apoptosis. We report that unlike other pro-apoptotic proteins, Bim contains two distinct binding sites for the anti-apoptotic proteins Bcl-XL and Bcl-2. These include the BH3 sequence shared with other pro-apoptotic proteins and an unexpected sequence located near the Bim carboxyl-terminus (residues 181–192). Using automated Fluorescence Lifetime Imaging Microscopy - Fluorescence Resonance Energy Transfer (FLIM-FRET) we show that the two binding interfaces enable Bim to double-bolt lock Bcl-XL and Bcl-2 in complexes resistant to displacement by BH3-mimetic drugs currently in use or being evaluated for cancer therapy. Quantifying in live cells the contributions of individual amino acids revealed that residue L185 previously thought involved in binding Bim to membranes, instead contributes to binding to anti-apoptotic proteins. This double-bolt lock mechanism has profound implications for the utility of BH3-mimetics as drugs. ​

scholarly journals Structural rearrangement of the intracellular domains during AMPA receptor activation

2016 ◽  
Vol 113 (27) ◽  
pp. E3950-E3959 ◽  
Author(s):  
Linda G. Zachariassen ◽  
Ljudmila Katchan ◽  
Anna G. Jensen ◽  
Darryl S. Pickering ◽  
Andrew J. R. Plested ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Resonance Energy ◽  
Normal Activity ◽  
Receptor Activation ◽  
Hek293 Cells ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Receptor Function ◽  
Loop Region ◽  
Intracellular Domains

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact.

scholarly journals Quantitative FRET-FLIM-BlaM to Assess the Extent of HIV-1 Fusion in Live Cells

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 206
Author(s):  
Irene Carlon-Andres ◽  
Sergi Padilla-Parra
Keyword(s):  
Single Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Novel Approach ◽  
Different Populations ◽  
Hiv Envelope ◽  
Hiv 1

The first steps of human immunodeficiency virus (HIV) infection go through the engagement of HIV envelope (Env) with CD4 and coreceptors (CXCR4 or CCR5) to mediate viral membrane fusion between the virus and the host. New approaches are still needed to better define both the molecular mechanistic underpinnings of this process but also the point of fusion and its kinetics. Here, we have developed a new method able to detect and quantify HIV-1 fusion in single live cells. We present a new approach that employs fluorescence lifetime imaging microscopy (FLIM) to detect Förster resonance energy transfer (FRET) when using the β-lactamase (BlaM) assay. This novel approach allows comparing different populations of single cells regardless the concentration of CCF2-AM FRET reporter in each cell, and more importantly, is able to determine the relative amount of viruses internalized per cell. We have applied this approach in both reporter TZM-bl cells and primary T cell lymphocytes.

scholarly journals Chromatin nanoscale compaction in live cells visualized by acceptor-donor ratio corrected FRET between DNA dyes

2019 ◽  
Author(s):  
Simone Pelicci ◽  
Alberto Diaspro ◽  
Luca Lanzanò
Keyword(s):  
Dna Damage ◽  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Donor And Acceptor ◽  
Dna Dyes

AbstractChromatin nanoscale architecture in live cells can be studied by Forster Resonance Energy Transfer (FRET) between fluorescently labeled chromatin components, such as histones. A higher degree of nanoscale compaction is detected as a higher FRET level, since this corresponds to a higher degree of proximity between donor and acceptor molecules. However, in such a system the stoichiometry of the donors and acceptors engaged in the FRET process is not well defined and, in principle, FRET variations could be caused by variations in the acceptor-donor ratio rather than distance. Here we show that a FRET value independent of the acceptor-donor ratio can be obtained by Fluorescence Lifetime Imaging (FLIM) detection of FRET combined with a normalization of the FRET level to a pixel-wise estimation of the acceptor-donor ratio. We use this method to study FRET between two DNA binding dyes staining the nuclei of live cells. We show that acceptor-donor ratio corrected FRET imaging reveals variations of nanoscale compaction in different chromatin environments. As an application, we monitor the rearrangement of chromatin in response to laser-induced micro-irradiation and reveal that DNA is rapidly decompacted, at the nanoscale, in response to DNA damage induction.

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.

Imaging protein–protein interactions in cell motility using fluorescence resonance energy transfer (FRET)

2004 ◽  
Vol 32 (3) ◽  
pp. 431-433 ◽  
Author(s):  
M. Parsons ◽  
B. Vojnovic ◽  
S. Ameer-Beg
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Intact Cell ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Fluorescence Resonance

Protein–protein interactions and signal transduction pathways have traditionally been analysed using biochemical techniques or standard microscopy. Although invaluable in the delineation of protein hierarchy, these methods do not provide information on the true spatial and temporal nature of complex formation within the intact cell. Recent advances in microscopy have allowed the development of new methods to analyse protein–protein interactions at very high resolution in both fixed and live cells. The present paper provides a brief overview of using fluorescence resonance energy transfer to analyse directly molecular interactions and conformational changes in various proteins involved in the regulation of cell adhesion and motility.

scholarly journals In vivomonitoring of plant small GTPase activation using a Förster resonance energy transfer biosensor

2018 ◽  
Author(s):  
Hann Ling Wong ◽  
Akira Akamatsu ◽  
Qiong Wang ◽  
Masayuki Higuchi ◽  
Tomonori Matsuda ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Plant Cells ◽  
Resonance Energy ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Living Plant ◽  
Förster Resonance

ABSTRACTSmall GTPases act as molecular switches that regulate various plant responses such as disease resistance, pollen tube growth, root hair development, cell wall patterning and hormone responses. Thus, to monitor their activation status within plant cells is believed to be the key step in understanding their roles. We have established a plant version of a Förster resonance energy transfer (FRET) probe called Ras and interacting protein chimeric unit (Raichu) that can successfully monitor activation of the rice small GTPase OsRac1 during various defence responses in rice cells. Here, we describe the protocol for visualizing spatiotemporal activity of plant Rac/ROP GTPase in living plant cells, transfection of rice protoplasts withRaichu-OsRac1and acquisition of FRET images. Our protocol should be widely adaptable for monitoring activation for other plant small GTPases and for other FRET sensors in various plant cells.

A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

2020 ◽  
Author(s):  
Brittany Benlian ◽  
Pavel Klier ◽  
Kayli Martinez ◽  
Marie Schwinn ◽  
Thomas Kirkland ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Membrane Potential ◽  
Small Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Potential Oscillations ◽  
Excitation Light ◽  
Voltage Imaging ◽  
Induced Pluripotent

<p>We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This <u>Q</u>uenching <u>B</u>ioluminescent V<u>olt</u>age Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.</p> <p> </p>

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