scholarly journals Analysis of GPI-Anchored Receptor Distribution and Dynamics in Live Cells by Tag-Mediated Enzymatic Labeling and FRET

2020 ◽  
Vol 3 (2) ◽  
pp. 33
Author(s):  
Maria N. Balatskaya ◽  
Alexandra I. Baglay ◽  
Yury P. Rubtsov ◽  
George V. Sharonov
Keyword(s):  
Cellular Response ◽  
Fluorescent Proteins ◽  
Cluster Formation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Labeling ◽  
Molar Ratio ◽  
Live Cells ◽  
Fluorescent Substrate ◽  
Receptor Distribution

The analysis of glycosylphosphatidylinositol (GPI)-anchored receptor distribution and dynamics in live cells is challenging, because their clusters exhibit subdiffraction-limited sizes and are highly dynamic. However, the cellular response depends on the GPI-anchored receptor clusters’ distribution and dynamics. Here, we compare three approaches to GPI-anchored receptor labeling (with antibodies, fluorescent proteins, and enzymatically modified small peptide tags) and use several variants of Förster resonance energy transfer (FRET) detection by confocal microscopy and flow cytometry in order to obtain insight into the distribution and the ligand-induced dynamics of GPI-anchored receptors. We found that the enzyme-mediated site-specific fluorescence labeling of T-cadherin modified with a short peptide tag (12 residues in length) have several advantages over labeling by fluorescent proteins or antibodies, including (i) the minimized distortion of the protein’s properties, (ii) the possibility to use a cell-impermeable fluorescent substrate that allows for selective labeling of surface-exposed proteins in live cells, and (iii) superior control of the donor to acceptor molar ratio. We successfully detected the FRET of GPI-anchored receptors, T-cadherin, and ephrin-A1, without ligands, and showed in real time that adiponectin induces stable T-cadherin cluster formation. In this paper (which is complementary to our recent research (Balatskaya et al., 2019)), we present the practical aspects of labeling and the heteroFRET measurements of GPI-anchored receptors to study their dynamics on a plasma membrane in live cells.

scholarly journals A FRET sensor of C-terminal movement reveals VRAC activation by plasma membrane DAG signaling rather than ionic strength

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Benjamin König ◽  
Yuchen Hao ◽  
Sophia Schwartz ◽  
Andrew JR Plested ◽  
Tobias Stauber
Keyword(s):  
Plasma Membrane ◽  
Ionic Strength ◽  
Patch Clamp ◽  
Fluorescent Proteins ◽  
Cell Volume Regulation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Activation Mechanism ◽  
Live Cells ◽  
Whole Cell Patch Clamp

Volume-regulated anion channels (VRACs) are central to cell volume regulation. Recently identified as hetero-hexamers formed by LRRC8 proteins, their activation mechanism remains elusive. Here, we measured Förster resonance energy transfer (FRET) between fluorescent proteins fused to the C-termini of LRRC8 subunits. Inter-subunit FRET from LRRC8 complexes tracked VRAC activation. With patch-clamp fluorometry, we confirmed that the cytoplasmic domains rearrange during VRAC opening. With these FRET reporters, we determined VRAC activation, non-invasively, in live cells and their subcompartments. Reduced intracellular ionic strength did not directly activate VRACs, and VRACs were not activated on endomembranes. Instead, pharmacological manipulation of diacylglycerol (DAG), and protein kinase D (PKD) activity, activated or inhibited plasma membrane-localized VRACs. Finally, we resolved previous contradictory reports concerning VRAC activation, using FRET to detect robust activation by PMA that was absent during whole-cell patch clamp. Overall, non-invasive VRAC measurement by FRET is an essential tool for unraveling its activation mechanism.

Fluorescent proteins and fluorescence resonance energy transfer (FRET) as tools in signaling research

2007 ◽  
Vol 97 (03) ◽  
pp. 378-384 ◽  
Author(s):  
Andreas Birbach ◽  
Johannes Schmid
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Proteins ◽  
Flow Analysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Signaling Dynamics ◽  
Biochemical Assays ◽  
Fluorescence Resonance

SummaryThe advent of fluorescent proteins has revolutionized signaling research, shifting focus from biochemical assays to analysis of live cells, organized tissues and even entire organisms. Modern applications of fluorescent proteins go beyond their use as specific markers of cells or tissues, allowing the researcher to visualize intracellular translocations as well as biochemical reactions. In this mini-review, we summarize the properties of a variety of fluorescent proteins, their detection using fluorescence microscopy and flow analysis, as well as their basic and more advanced applications, including fluorescence resonance energy transfer (FRET) to study signaling dynamics.

scholarly journals Lighting Up the Force: Investigating Mechanisms of Mechanotransduction Using Fluorescent Tension Probes

2015 ◽  
Vol 35 (15) ◽  
pp. 2570-2582 ◽  
Author(s):  
Carol Jurchenko ◽  
Khalid S. Salaita
Keyword(s):  
Cellular Response ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Physical Nature ◽  
Fine Tuning ◽  
Cell Stiffness ◽  
Live Cells ◽  
Physical Cues ◽  
Multicellular Organisms ◽  
Dynamic Cycle

The ability of cells to sense the physical nature of their surroundings is critical to the survival of multicellular organisms. Cellular response to physical cues from adjacent cells and the extracellular matrix leads to a dynamic cycle in which cells respond by remodeling their local microenvironment, fine-tuning cell stiffness, polarity, and shape. Mechanical regulation is important in cellular development, normal morphogenesis, and wound healing. The mechanisms by which these finely balanced mechanotransduction events occur, however, are not well understood. In large part, this is due to the limited availability of tools to study molecular mechanotransduction events in live cells. Several classes of molecular tension probes have been recently developed which are rapidly transforming the study of mechanotransduction. Molecular tension probes are primarily based on fluorescence resonance energy transfer (FRET) and report on piconewton scale tension events in live cells. In this minireview, we describe the two main classes of tension probes, genetically encoded tension sensors and immobilized tension sensors, and discuss the advantages and limitations of each type. We discuss future opportunities to address major biological questions and outline the challenges facing the next generation of molecular tension probes.

Distribution of lipid raft markers in live cells

2004 ◽  
Vol 32 (5) ◽  
pp. 673-675 ◽  
Author(s):  
O.O. Glebov ◽  
B.J. Nichols
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Interactions ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Receptor ◽  
Specific Protein ◽  
Live Cells ◽  
Jurkat T Cells

GPI (glycosylphosphatidylinositol)-anchored proteins are characteristic components of biochemically defined lipid rafts. Rafts may be involved in T-cell stimulation, but it is not clear whether molecules involved in TCR (T-cell receptor) signalling are partitioned to T-cell synapses through raft microdomains or through specific protein–protein interactions. We have used FRET (fluorescence resonance energy transfer) analysis to study the distribution of GPI-anchored fluorescent proteins in the plasma membrane of live cells. Multiple criteria suggested that FRET between different GPI-anchored fluorescent proteins in COS-7 or unstimulated Jurkat T-cells is generated by a random, unclustered distribution. Stimulation of TCR signalling in Jurkat T-cells by beads coated with antibodies against TCR subunits resulted in localized increases in fluorescence of raft markers. However, measurements of FRET and ratio imaging showed that there was no detectable clustering and no overall enrichment of raft markers in these regions.

Quantum Dot Bioconjugates as Energy Donors in Fluorescence Resonance Energy Transfer Assays

2003 ◽  
Vol 773 ◽  
Author(s):  
Aaron R. Clapp ◽  
Igor L. Medintz ◽  
J. Matthew Mauro ◽  
Hedi Mattoussi
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Genetically Engineered ◽  
Molar Ratio ◽  
Binding Assays ◽  
Specific Sequence ◽  
Donor Acceptor

AbstractLuminescent CdSe-ZnS core-shell quantum dot (QD) bioconjugates were used as energy donors in fluorescent resonance energy transfer (FRET) binding assays. The QDs were coated with saturating amounts of genetically engineered maltose binding protein (MBP) using a noncovalent immobilization process, and Cy3 organic dyes covalently attached at a specific sequence to MBP were used as energy acceptor molecules. Energy transfer efficiency was measured as a function of the MBP-Cy3/QD molar ratio for two different donor fluorescence emissions (different QD core sizes). Apparent donor-acceptor distances were determined from these FRET studies, and the measured distances are consistent with QD-protein conjugate dimensions previously determined from structural studies.

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>

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>

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 GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET

2009 ◽  
Vol 134 (6) ◽  
pp. 489-521 ◽  
Author(s):  
Fraser J. Moss ◽  
P.I. Imoukhuede ◽  
Kimberly Scott ◽  
Jia Hu ◽  
Joanna L. Jankowsky ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Fluorescent Proteins ◽  
Resonance Energy ◽  
High Order ◽  
Live Cells ◽  
Gaba Uptake ◽  
Cell Periphery ◽  
Wild Type ◽  
Gaba Transporter ◽  
Amplitude Component

The mouse γ-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [3H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [3H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ∼30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst.

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