scholarly journals Generation and Characterization of a New FRET-Based Ca2+ Sensor Targeted to the Nucleus

2021 ◽  
Vol 22 (18) ◽  
pp. 9945
Author(s):  
Luisa Galla ◽  
Nicola Vajente ◽  
Diana Pendin ◽  
Paola Pizzo ◽  
Tullio Pozzan ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cellular Processes ◽  
A Cell ◽  
Dynamic Concentration ◽  
Subcellular Compartmentalization

Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented.

scholarly journals Activation Function 1 of Glucocorticoid Receptor Binds TATA-Binding Protein in Vitro and in Vivo

2006 ◽  
Vol 20 (6) ◽  
pp. 1218-1230 ◽  
Author(s):  
Alicja J. Copik ◽  
M. Scott Webb ◽  
Aaron L. Miller ◽  
Yongxin Wang ◽  
Raj Kumar ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Glucocorticoid Receptor ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Activation Function ◽  
Tata Binding Protein

Abstract The mechanism through which the glucocorticoid receptor (GR) stimulates transcription is still unclear, although it is clear that the GR affects assembly of the transcriptional machinery. The binding of the TATA-binding protein (TBP) to the TATA-box is accepted as essential in this process. It is known that the GR can interact in vitro with TBP, but the direct interaction of TBP with GR has not been previously characterized quantitatively and has not been appreciated as an important step in assembling the transcriptional complex. Herein, we demonstrate that the TBP-GR interaction is functionally significant by characterizing the association of TBP and GR in vitro by a combination of techniques and confirming the role of this interaction in vivo. Combined analysis, using native gel electrophoresis, sedimentation equilibrium, and isothermal microcalorimetry titrations, characterize the stoichiometry, affinity, and thermodynamics of the TBP-GR interaction. TBP binds recombinant GR activation function 1 (AF1) with a 1:2 stoichiometry and a dissociation constant in the nanomolar range. In vivo fluorescence resonance energy transfer experiments, using fluorescently labeled TBP and various GR constructs, transiently transfected into CV-1 cells, show GR-TBP interactions, dependent on AF1. AF1-deletion variants showed fluorescence resonance energy transfer efficiencies on the level of coexpressed cyan fluorescent protein and yellow fluorescent protein, indicating that the interaction is dependent on AF1 domain. To demonstrate the functional role of the in vivo GR-TBP interaction, increased amounts of TBP expressed in vivo stimulated expression of GR-driven reporters and endogenous genes, and the effect was also specifically dependent on AF1.

scholarly journals Bacterial dynamin-like protein DynA mediates lipid and content mixing and shows phospholipid specificity

2018 ◽  
Author(s):  
Lijun Guo ◽  
Marc Bramkamp
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Stress ◽  
Gtp Hydrolysis ◽  
Cellular Processes ◽  
Organelle Division ◽  
Membrane Tethering ◽  
Hydrolysis Of

ABSTRACTThe dynamins family of GTPases is involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. The GTP hydrolysis cycle of dynamin translates to a conformational change in the protein structure, which forces the underlying lipid layer into an energetically unstable conformation that promotes membrane rearrangements. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. In recent years, our group has reported that the dynamin-like protein DynA from Bacillus subtilis mediates nucleotide-independent membrane tethering in vitro and contributes to the innate immunity of bacteria against membrane stress and phage infection. However, so far the mechanism of membrane stress response and the role of GTP hydrolysis remain unclear. Here, we employed content mixing and lipid mixing assays in reconstituted systems to study if the dynamin-like protein DynA from B. subtilis induces membrane full fusion, and further test the possibility that GTP hydrolysis of DynA may act on the fusion-through-hemifusion pathway. Our results based on fluorescence resonance energy transfer (FRET) indicated that DynA could induce aqueous content mixing even in absence of GTP. Moreover, DynA-induced membrane fusion in vitro is a thermo-promoted slow response. Surprisingly, digestion of protein mediated an instantl rise of content exchange, supporting the assumption that disassembly of DynA is the fundamental power for fusion-through-hemifusion.

scholarly journals A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy

2015 ◽  
Vol 309 (11) ◽  
pp. C724-C735 ◽  
Author(s):  
Wen Tao ◽  
Michael Rubart ◽  
Jennifer Ryan ◽  
Xiao Xiao ◽  
Chunping Qiao ◽  
...  
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Practical Method ◽  
Two Photon Microscopy ◽  
Cellular Processes ◽  
Two Photon ◽  
Photon Excitation ◽  
Wide Range

The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes.

scholarly journals A Fluorescent Protein-Based Biological Screen of Proteinase Activity

2010 ◽  
Vol 15 (2) ◽  
pp. 224-229 ◽  
Author(s):  
Carly Huitema ◽  
Lindsay D. Eltis
Keyword(s):  
Fluorescent Protein ◽  
Hepatitis A Virus ◽  
Cysteine Proteinase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Proteinase Activity ◽  
General Utility ◽  
Small Molecule Libraries ◽  
A Cell ◽  
Green Fluorescent

A cell-based fluorescent protein reporter assay for proteinase activity amenable to high-throughput applications was developed. This assay is based on Förster resonance energy transfer (FRET) between 2 variants of the green fluorescent protein connected by a short cleavable linker and expressed in Escherichia coli as tagged proteins. A library to assay proteinase specificity was generated by randomizing a portion of the linker using PCR. The library could be grown in microplates, allowing cells to be lysed in situ and substrate cleavage to be monitored through loss of FRET signal using a plate reader. Progress curves were generated to estimate cleavage efficiency, facilitating the identification of well-cleaved substrates. The polyhistidine-tagged fluorescent substrates could then be purified and used for further characterization. To establish the general utility of the screen, it was used to demonstrate that the cysteine proteinase of the hepatitis A virus, 3Cpro, prefers Ile, Val, or Leu at the P4 position of the cleavage sequence and Gly, Ser, or Ala at the P′1 position. The assay can also be used to screen small-molecule libraries for inhibitors.

scholarly journals Nicotine up-regulates α4β2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning

2010 ◽  
Vol 137 (1) ◽  
pp. 59-79 ◽  
Author(s):  
Rahul Srinivasan ◽  
Rigo Pantoja ◽  
Fraser J. Moss ◽  
Elisha D.W. Mackey ◽  
Cagdas D. Son ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Pharmacological Chaperone ◽  
Clinical Literature ◽  
Er Exit Sites ◽  
A Cell ◽  
Er Export

The up-regulation of α4β2* nicotinic acetylcholine receptors (nAChRs) by chronic nicotine is a cell-delimited process and may be necessary and sufficient for the initial events of nicotine dependence. Clinical literature documents an inverse relationship between a person’s history of tobacco use and his or her susceptibility to Parkinson’s disease; this may also result from up-regulation. This study visualizes and quantifies the subcellular mechanisms involved in nicotine-induced nAChR up-regulation by using transfected fluorescent protein (FP)-tagged α4 nAChR subunits and an FP-tagged Sec24D endoplasmic reticulum (ER) exit site marker. Total internal reflection fluorescence microscopy shows that nicotine (0.1 µM for 48 h) up-regulates α4β2 nAChRs at the plasma membrane (PM), despite increasing the fraction of α4β2 nAChRs that remain in near-PM ER. Pixel-resolved normalized Förster resonance energy transfer microscopy between α4-FP subunits shows that nicotine stabilizes the (α4)2(β2)3 stoichiometry before the nAChRs reach the trans-Golgi apparatus. Nicotine also induces the formation of additional ER exit sites (ERES). To aid in the mechanistic analysis of these phenomena, we generated a β2enhanced-ER-export mutant subunit that mimics two regions of the β4 subunit sequence: the presence of an ER export motif and the absence of an ER retention/retrieval motif. The α4β2enhanced-ER-export nAChR resembles nicotine-exposed nAChRs with regard to stoichiometry, intracellular mobility, ERES enhancement, and PM localization. Nicotine produces only small additional PM up-regulation of α4β2enhanced-ER-export receptors. The experimental data are simulated with a model incorporating two mechanisms: (1) nicotine acts as a stabilizing pharmacological chaperone for nascent α4β2 nAChRs in the ER, eventually increasing PM receptors despite a bottleneck(s) in ER export; and (2) removal of the bottleneck (e.g., by expression of the β2enhanced-ER-export subunit) is sufficient to increase PM nAChR numbers, even without nicotine. The data also suggest that pharmacological chaperoning of nAChRs by nicotine can alter the physiology of ER processes.

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 Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomomi Kaku ◽  
Kazunori Sugiura ◽  
Tetsuyuki Entani ◽  
Kenji Osabe ◽  
Takeharu Nagai
Keyword(s):  
Energy Transfer ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Color Change ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Bacterial Luciferase ◽  
Bacterial Bioluminescence

AbstractUsing the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.

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